CA2957707C - Mutations in iron-sulfur cluster proteins that improve xylose utilization - Google Patents

Abstract

There is provided an engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a polypeptide having xylose isomerase activity, and methods of their use thereof.

Description

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MUTATIONS IN IRON-SULFUR CLUSTER PROTEINS THAT IMPROVE XYLOSE UTILIZATION
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
10001] This invention was funded, in part, by the United States government under a grant with the Department of Energy. Office of Energy Efficiency and Renewable Energy. Bioencrgy Technologies Office. Award No. DE-FC36-015G018103 to Masco= and FWP-IICEEEtr)07 to Oak Ridge National Laboratory. This invention was also funded, in part, by the Biocnergy Science Center, Oak Ridge National Laboratory. a 1.1.S. Depanment of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research. under contract DE-PS02-06ER6430-1. The government has certain rights in the invention.
FIELD OF THE INVENTION
100021 The field of the invention generally relates to engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a poly peptide tuning xylose isomerase activity; and methods of fermenting cellulosic biomass to produce biofuels, including ethanol, BACKUROUND OF THE INVENTION
100031 Saocharomyces ceresisiac is the primary biocatalyst used in the commercial production of "first generation" fuel ethanol from sugar based siibstrates such as corn, sugarcane, and sugarbeet. Second generation ethanol production, also known as cellulosic ethanol production, extends the carbohydrate source to more complex polysaccharides, such as cellulose and hernicellidose, which make up a significant portion of most plant cell walls and therefore most plant material.
100041 Feedstocks commercially considered for second generation ethanol production include wood.
agriculture residues such as coni stover and wheat straw, sugarcane bagasse and purpose grown materials such as switchgrass. The cellulose and hemicellose must be hydrolyzed to monomeric sugars before fermentation using either mechanical/chemical means and/or enzymatic hydrolysis. The liberated monomeric sugars include glucose. xylose, galactose. nnumose. and arabinose with glucose and xylose constituting more than 75% of the monomeric sugars in most feedstocks. For cellulosic ethanol production to be economically viable and compete with first generation ethaaol, the biocatalyst must be able to convert the majority, if not all, of the available sugars into ethanol.
100051 S. cerevisiae is the preferred organism for first generation ethanol production due to its robustness, high yield, and many years of safe use. However, naturally occurring S. ccrevisiae is unable to ferment ,µ) lose into ethanol. For S. cerevisiae to be a viable biocatalyst for second generation ethanol production, it must be able to ferment xylose.
100061 There are two metabolic pathways of xylose fermentation that have been demonstrated in S. cerevisiae.
The pathways differ primarily in the conversion of xylose to xylulose. In the first pathway. the XR-XDH
pathway. a xylose reductase (XR) converts xylose to xylitol, which is subsequently convened to xylulose by a xylitol dehydrogenase (XDH). The XR and XDH enzyme pairs tested to date differ in required cofactor. NADH
and NADPH. leading to difficulties achieving redox balance. The second commonly tried pathway converts xylose directly to xylulose using a xylose isomerase (XI) with no redox cofactor requirements. Xis from both bacterial and fungal systems have beet) successfully utilized in S.
cerevisiae. Both pathways utilize the same downstreani metabolic engineering: up regulation of the native xylulose kinase (XKS I ) and four genes of the pentose phosphate pathway. specifically ribulose-phosphate 3-epimerase (RPE
I). ribose-5-phosphate ketol-

2 a isomerase (RKI1), transaldolase (TALI), and transketolase (TKL1) (Figure 1).
Use of the XI pathway also commonly entails deletion of the native aldose reductase gene (GRE3) to eliminate product lost to xylitol formation.
[0007] Xylose isomerases are known to have several metal ion binding sites, which allows XIs to bind metal ions such as manganese, cobalt, and magnesium. See, e.g., Chang et al., "Crystal Structures of Thermostable Xylose Isomerases from Thermus caldophilus and Thermus thermophilus: Possible Structural Determinants of Thermostability," J.
Mol. Biol 288:623-34 (1999). There is some indication that XIs may also bind iron cations (Fe+), but Fe+ is usually not the preferred or optimal divalent cation. However, intracellular iron regulation and metabolism is known to be a critical function for eukaryotic cells due to iron's role as a redox-active protein cofactor. See, e.g., Outten and Albetel, "Iron sensing and regulation in Saccharomyces cerevisiae: Ironing out the mechanistic details," Curr. Op.
Microbiol. 16:662-68 (2013). Intracellular iron levels are primarily controlled by the iron-sensing transcriptional activators Aft 1 and Aft2 in S. cerevisiae. Iron-sulfur (Fe/S) clusters are essential for transcriptional control by Aft 1/2 and Yap5 during iron sufficiency. Under sufficient iron levels, Fe/S clusters are synthesized in the mitochondria through the integration of iron, sulfur, and redox control pathways. The Fe/S clusters interact with Grx3, Grx4, Fral, and Fra2 to inactivate Aft1/2, leading to down regulation of Aft1/2 target genes. Fe/S clusters also are known to activate the expression of Yap5 target genes, including CCC1. Ccc 1 stimulates the import of iron and its sequestration in the vacuole.
BRIEF SUMMARY OF THE INVENTION
[0008] Aspects of the invention are directed to engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a polypeptide having xylose isomerase activity, and methods of their use are described herein.
[0009] In some embodiments, the host cell heterologously expresses one or more polypeptides capable of converting xylose to xylulose. In some embodiments, the one or more heterologously expressed polypeptide is a xylose isomerase. In some embodiments, the heterologously expressed polypeptide is a naturally occurring polypeptide.
In some embodiments, the heterologously expressed polypeptide is recombinant. In some embodiments, the heterologously expressed polypeptide is a chimeric polypeptide. In some i 2a . .
embodiments, the chimeric polypeptide is as described in the related provisional application US62/035,752 filed on August 11, 2014.
[0010] In some embodiments of the present invention, the heterologously expressed polypeptide has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and/or 27. In some embodiments, the heterologously expressed polypeptide has an amino acid sequence of SEQ ID
NOs: 1,

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In some embodiments of the present invention, the heterologously expressed polypeptide has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 35, 37, 39, and/or 41,. In some embodiments, the heterologously expressed polypeptide has an amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 35, 37, 39, or 41.
[0011] In some embodiments, the heterologously expressed polypeptide is encoded by a polynucleotide sequence having al least 50%. at least 55%, at least 60%, at least 65%, at least 70%, at least 75%. at least 760/u, at least 77%, at least 78%, at least 79%, at least 80%,.. at least 81%, at least 82%, at least 83%, at least 84%, at least 115%. at least 86%, at least 87%, at least 88%. at least 89%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 93%, at least 96%, at least 97%. at least 98%, or at least 99% sequence identity with any one of SEQ NOs: 2, 4, 6. 8, 10, 12, 14. 16, 18, 20. 22, 24, 26. and/or 28.
In sonic embodiments, the licterologonsly espressed polypeptide is encoded by a oolymieleotide sequence of SEQ tD NOs: 2, 4, 6. 8. 10, 12. 14. 16, 18. 20, 22. 24. 26. or 28. In some embodiments, the beterologously expressed polypeptide is encoded by a polynueleotide sequence having at least 50%, at least 55%, at least 60%.
at least 65% at least 70%. at least.
75%. at least 76%, at least 77%. at least 184 at least '79%. at least 80%. at least 81% at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 37%. at least 88% at least 89%. at least 90%, at least 91%. at least 924, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%
sequence identity with any one of SEQ ID NOs: 2, 4. 6. 8, 10, 12, 14, 16, 18, 20. 22, 24, 26. 28, 36. 38, 40, and/or 42, In some embodiments, the beterologously expressed polypeptide is encoded by a polynucleotide sequence of SEQ ID NOs: 2. 4, 6, 8, 10, 12, 14, 16, IS, 20. 22, 24. 26, 28, 36. 38, 40. or 42. In some embodiments, the polynucleotide sequence is contained in a vector.
100121 In some embodiments, a host cell is engineered to express one or more of the chimeric polypeptides. In some embodiments. the host cell is a yeast cell, e.g. a S. cerevisiae cell. In sonic embodiments the host cell is further modified to have mutations affecting at least one gene encoding a protein involved in the peniose phosphate pathway, lit sonic embodiments, the host cell has at least one mutation that increases the expression or causes the up-regulation of XKS1. RKI 1, RPE I TKL I. and/or TALI. In some embodiments. the host cell has a modification of one or more aldose reductase genes. In sonic embodiments, the tildose reductase gene is GRE3.
In some embodiments, the host cell has a deletion or disruption of all or part of the endogenous GRE3 gene, hi sonic embodiments, the aldose reductase gene is YPR1. In some embodiments, the host cell has a deletion or disruption of all or part of the endogenous YPR.1 gene. In some embodiments, the host cell has a deletion or disruption of all or part of both the endogenous GRE3 gene and the endogenous YPR.I gem. In some embodiments. the host cell has a modification of PGM1 (phosphoglueomutase 1) and/or PGM2. In some embodiments, the host cell overespresses PGM.1 and/or PGM2. In some embodiments, the host cell has increased levels of Pgml and/or Pg.m2 polypeptide and/or niRNA relative to a comparable host cell lacking a modification of PGM I and/or PGM2, 100131 In some embodiments, the host cell comprises a deletion or disruption of one or more endogenous enzymes that function to produce glycerol and/or regulate glycerol synthesis.
In some embodiments, the host cell moduces less glycerol than a control recombinant microorganism without deletion or disruption of said one or more endogenous enzymes that function to produce glycerol and/or regulate glycerol synthesis. In sonic embodiments, the one or morc endogenous enzymes that function to produce glycerol are encoded by a GPM
polynticleotide, a GPD2 poly nucleotide, or both a GPD1 polyaucleotidc and a GPD2 polynucicolide, In some embodiments. one or both of the endogenous GPO! and/or GPD2 genes arc modified by mutation or deletion. In some embodiments, the host cell comprises a heterologous ADHE sequence. In some embodiments, the heterologons ADHE is from Bilidobacteritun adolescentis. In some embodiments the native STL1 gene is upregubted by either modifying the promoter of the native copies or by introducing additional copies of STL I.
In some embodiments the host cell comprises an ortholog of the native STL1õ in sonic embodiments the native

4 ACS2 gene is upregulated by either modifying the promoter of the native copies or by introducing additional copies of ACS2. In some embodiments the host cell comprises an ortholog of the native ACS2 or ACS1 gene.
[0014] In some embodiments, the host cell comprises one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism. In some embodiments, the host cell comprises one or more mutations in one or more endogenous genes encoding an iron uptake protein, iron utilization protein, and/or an iron/sulfur (Fe/S) cluster biosynthesis protein. In some embodiments, the host cell comprises one or more mutations in one or more endogenous genes encoding a polypeptide affecting iron metabolism or Fe/S cluster biosynthesis. In some embodiments, the host cell is a recombinant yeast cell. In some embodiments, the recombinant yeast cell comprises one or more mutations in one or more of an endogenous gene selected from the group ISUl, YFH1, NFS1, AFT1, AFT2, YAPS, FRAI, FRA2, GREX3, GREX4, CCC1, and combinations thereof. In some embodiments, the recombinant yeast cell comprises one or more mutations in one or more of an endogenous gene which is homologous to one or more of an S. cerevisiae gene selected from the group ISU1, YFH1, NFS I, AFT1, AFT2, YAPS, FRA1, FRA2, GREX3, GREX4, and CCC1. In some embodiments, the recombinant yeast cell comprises a mutation in the endogenous AFT1 or AFT2 gene that results in iron-independent activation of the iron regulon such as the AFT1-1up or AFT2-lup alleles (Rutherford et al., "Aftlp and Aft2p mediate iron-responsive gene expression in yeast through related promoter elements," JBC 278(30):27636-43 (2003)). In some embodiments, the recombinant yeast cell comprises a deletion or disruption of YAPS
and/or CCC1. In some embodiments, the recombinant yeast cell comprises a deletion or disruption of YAPS and/or CCC1 and/or a mutation in the endogenous AFT1 or gene that results in iron-independent activation of the iron regulon such as the AFT1-lup or AFT2-1up alleles.
[0015] In some embodiments, the host cell comprises one or more mutations in the endogenous ISU I gene that results in a polypeptide comprising at least one amino acid substitution selected from the group consisting of D71N, D71G, and 598F, wherein the position of the substitution is relative to the amino acid positions of SEQ ID
NO:29. In some embodiments, the host cell comprises one or more mutations in the endogenous 4a YFH1 gene that results in a polypeptide comprising a T163P substitution, wherein the position of the substitution is relative to the amino acid positions of SEQ ID
NO:31. In some embodiments, the host cell comprises one or more mutations in the endogenous NFS1 gene that results in a polypeptide comprising at least one amino acid substitution selected from the group consisting of LI 15W and E458D, wherein the position of the substitution is relative to the amino acid positions of SEQ ID NO:33.
[0016] In some embodiments, the host cell has a modification of PGM1 (phosphoglucomutase 1) and/or PGM2, as described in the related provisional application filed on August 11, 2014. In some embodiments, the host cell overexpresses PGM1 and/or PGM2. In some embodiments, the host cell has increased levels of Pgm 1 and/or Pgm2 polypeptide and/or mRNA relative to a comparable host cell lacking a modification of PGM1 and/or PGM2.
[0017] In some embodiments, the host cell expresses one or more heterologous genes encoding a protein that is associated with iron metabolism. In some embodiments, the heterologous gene confers on the recombinant yeast cell an increased ability to utilize xylose as compared to a similar yeast cell lacking the heterologous gene. In some embodiments, the heterologous gene is AFT1, AFT2, and/or an orthologue thereof. In some embodiments, the heterologous gene encodes a polypeptide having iron transport activity. In some embodiments, the heterologous gene encodes a protein that increases the activity and/or expression of Aft 1 and/or Aft2. In some embodiments, the heterologous gene is a target of Aft! and/or Aft2. In some embodiments, the heterologous gene is constitutively expressed. In some embodiments, the heterologous gene is overexpressed. In some embodiments, the heterologous gene encodes a protein that suppresses a gene or protein that suppresses Aft! and/or Aft2 activity and/or expression. In some embodiments, the heterologous gene encodes a protein that suppresses a gene or protein that suppresses the activity and/or expression of one or more downstream targets of Aft] and/or Aft2.
100181 In some embodiments, a yeast strain is used as the host cell. In some embodiments, the background of the yeast strain is an industrial yeast strain. One having ordinary skill in the art would be aware of many potential known yeast strains that can be modified according to the present invention, and this invention contemplates all such potential background yeast strains.
1001911n some embodiments of the invention, the recombinant host cell is used to produce a fermentation product from a cellulosic or lignocellulosic material. In some embodiments, the fermentation product is ethanol, lactic acid, 3-hydroxy-propionic acid, hydrogen, butyric acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, acetone, isopropyl alcohol, butanol, a p-lactam, an antibiotic, a cephalosporin, or a combination thereof In some embodiments, the cellulosic or lignocellulosic material is insoluble cellulose, crystalline cellulose, pretreated hardwood, paper sludge, pretreated corn stover, pretreated sugar cane bagasse, pretreated corn cobs, pretreated switchgrass, pretreated municipal solid waste, pretreated distiller's dried grains, pretreated wheat straw, corn fiber, agave, or a combination thereof.
100201 One aspect of the invention is directed to a composition comprising a lignocellulosic material and a recombinant yeast host cell comprising one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism and at least one gene encoding a polypeptide having xylose isomerase activity. Another aspect of the invention is directed to a media supernatant generated by incubating a recombinant yeast host comprising one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism and at least one gene encoding a polypeptide having xylose isoincruse activity with a medium containing xylose as the only carbon source. In some embodiments, the medium comprises a cellulosic or lignocellulosic material. In some embodiments, the cellulosic or lignocellulosic material is insoluble cellulose, crystalline cellulose, pretreated hardwood, paper sludge, saw mill or paper mill discards, pretreated corn stover, pretreated sugar cane bagasse, pretreated corn cobs, pretreated switchgrass, pretreated municipal solid waste, pretreated distiller's dried grains, pretreated wheat straw, corn fiber, agave, or a combination thereof.
10020a] In yet another aspect, the present invention provides a recombinant yeast cell comprising (a) at least one heterologous gene encoding a protein associated with iron metabolism and/or one or more mutations in one or more endogenous gene encoding a protein associated with iron metabolism; and (b) at least one heterologous gene encoding a polypeptide having xylose isomerase activity; whereby the at least one heterologous gene encoding a protein associated with iron metabolism and/or the one or more mutations in one or more endogenous gene encoding a protein associated with iron metabolism results in an 5a increased ability to utilize xylose by the recombinant yeast cell as compared to a similar yeast cell lacking the at least one heterologous gene encoding a protein associated with iron metabolism and/or the one or more mutations in one or more endogenous gene encoding a protein associated with iron metabolism.
BRIEF DESCRIPTION OF THE DRAWINGS
10021] Figure 1 depicts a schematic representation of xylose fermentation in genetically engineered S.
cerevisiae.
10022] Figure 2 depicts a schematic representation of the role of Fe/S
clusters in intracellular iron metabolism. See Outten and Albetel, ''Iron sensing and regulation in Saccharomyces cerevisiae: Ironing out the mechanistic details," Curr. Op. Microbiol. 16:662-68 (2013).
100231 Figure 3 provides examples of the relative growth of xylose utilizing yeast strains (XUS) with various mutations in genes encoding proteins associated with intracellular iron metabolism, specifically YFH1 (Figure 3A), ISU1 (Figure 3B), and NFS1 (Figure 3C).
100241 Figure 4 provides examples of the relative growth of xylose utilizing yeast strains (XUS) with =

5 PCT/1132915/0561111

6 heterozygous and homozygous mutations in genes encoding proteins associaied with intracellular iron metabolism. specifically ISU I (Figure 4A) and 15U1 and YFH I (Figure 413), in two XUS stniins.
100251 Figure 5 provides examples of the relative growth of xy lose utilizing y east strains hcterologously expressing selected xy lose isomerase genes. including those from B.
thetaiotaomicron (BtXI). Piromyces (PiX1).
C. abercnsis (CaX1). P. mininicola (PrXI). P. distasonis (PdXI). XYM2. A
defectiva (AdXI).
Lachixxinaerobactiltim sabuneum (LOCI). Clostridium phytofennentans (CpXI).
and Lactobacillus xylostis (LxX1). The growth levels for of each x,ylose utilizing yeast stain arc show with (hashed bars) and without (solid bars) the T163P mutation of YFH I.
100261 Figure 6 provides examples of the relative growth of yeast cells hetenalogously expn....ssing selected xylose isomerases (chromosomally integrated) including those from CX355 =
chimeric xylosc isomerase 355 .
CX1224 = chimeric xylosc isomerase 1224, Ad = Abiotrophia defectiva. St =
Bactcriodcs thetaioatomicron. Pe = Piromyces. Ls = Lachnoanacrobaculum sciburreum with and %vithoui a mutation in YFH I. The growth levels for of each xylosc utilizing yeast strain are show with (Figure (,A) and Avithout (Figure 613) the T163P mutation of YFH I .
100271 Figure 7 provides examples of the relative growth of xylose utilizing yeast strains (XUS) with various mutations in genes encoding proteins associated with intracellular iron metabolism. specifically AFT I. and ccel.
10028] Figure 8 provides examples of the relative ethanol production of xylose utilizing yeast strains (XUS) grown in gliicose/xylosc media with and without iron addition 100291 Figure 0 provides eNninples of in vitm x) lose isomenise activity assay of Nylose utiliAng east strains (XUS).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions 1903411 Unless defined otherwise, all technical and scientific tenns used herein base the same meaning as commonly understood to one of ordinary skill in the art of microbial metabolic engineering. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions. exemplary methods, devices and materials are described herein.
190311 The embodimmu(s) described, and references in the specification to "one embodiment". "an embodiment". "an example embodiment", etc.. indicate that the embodiment(s) described can include a particular feature, structure, or characteristic. but every embodiment does not necessanly include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily.
referring to the same embodiment.
Further. w hen a particular feature. stmeture. or characteristic is described in connection with an embodiment, it is understood that it is within the knowledge of one skilled in the art to effect such feature. structure. or characteristic in connection with other embodiments whether or not explicitly described.
100321 The description of "a" or "an" item herein refers to a single item or multiple items. It is understood that herever embodiments arc described herein with the language "comprising."
otherwise analogous embodiments dcscnbcd in terms of "consisting of and/or "consisting essentially' of are also provided. Thus, for example, reference to "a polynucicotide" includes a plurality of such poly nucleotides and reference to "the microorganism" includes reference to one or more microorganisms, and so forth.
190331 A "fragment" refers to any portion of a nucleic or amino acid sequence that is less than the entire sequence. A fragment of a ancleoinic or an amino acid sequence can be an length of nucleotides or amino acids = CA 02957707 2017-02-03

7 that is less than the entire length of the cited sequence and more than two nucleotides or amino acids in length. In some embodiments. the fragment can be from a donor sequence.
100341 A "vector." e.g., a "plasmid" or "YAC" (yeast anificial chromosome) refers to an extrachromosomal element often carry lug one or more genes that are not part of the central metabolism of the cell, and can be in the form of a linear or circular double-stranded DNA molecule. Vectors and plasmids can be autonomously replicating sequences. genomc integrating sequences. phage or nucleotide sequences. linear, circular, or supercoiled. of a single- or double-stranded DNA or RNA. derived front any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' war:instated sequence into a cell.
100351 An "expression sector" is a vector that is capable of directing the expression of genes to which it is operably associated.
100361 The term "integrated" as used herein refers to genetic elements that are placed, through molecular biology techniques, into the genomc of a host cell. For example. genetic elements can be placed into the chromosomes of the host ecul as opposed to in a plasmid carried by the host cell. Methods for integnaing genetic elements into the genome of a host cell are well known in the art and include homologous recombination. In some embodiments, more than onc copy of the genetic elements are placed into the gem= of a host cell. In some embodiments. 2. 3.4, 5. 6. 7. 8, 9, 10, or more copies of the genetic elements are placed into the gnome of a host cell.
100371 The term "heterologons" when used in reference to a polynucleotide. a gene. a polypeptide. or an enzyme refers to a polynucleotide. gene, polypeptide. or an enzyme not normally found in the host organism.
"Hcterologous" also includes a native coding region. or portion thereof, that is removed front the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding natise gene. e.g.. not in its natural location in the organism's genome. The heterologons poly nucleotide or gene can be introduced into the host organism by. e.g..
gene transfer. A heterologous gene can include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Foreign genes can comprise native genes inserted into a non-native organism.
or chimeric genes. A heterologous poly nucleotide, gene. pial peptide. or an cozy me can be derived from any source. e.g.. cukaryotes. prokars otes. vimses. or sy mimic polynticleotide fragments. The term "hctcrologous" as used herein also refers to tin element of a vector. plasmid or host cell that is derived front a source other than the endogenous source. Thus, for example. a Iteterologous sequence could be a sequence that is derived from a different gene or plastind front the same host, from a different strain of host cell, or from an organism of a different taxonomic group (e.g_. different kingdom. phylum, class. order.
family, genus, or species, or an subgroup within one of these classifications). The term "heterologous" is also used synonymously herein with the term "exogenous." The term "hetcrologous expression" refers to the expression of a heterologous polynneleotide or gene by- a host.
100381 The term "domain" as used herein refers to a part of a molecule or structure that shares common physical or chemical features, for example hydrophobic. polar. globular, helical domains or properties. e.g.. a DNA binding domain or an ATP binding domain. Domains can be Identified by their homology to conserved structural or functional motifs. Examples of cellobiohydrolase (CBH) domains include the catalytic domain (CD)

8 and the cellulose binding domain (CBD).
[0039] A "nucleic acid," "polynucleotide," or "nucleic acid molecule" is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.
[00401 An "isolated nucleic acid molecule" or "isolated nucleic acid fragment"
refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine:
"RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA
molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA
and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA
molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences are described herein according to the normal convention of giving only the sequence in the 5' to 3 direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
[0041] A "gene" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a specific protein, including intervening sequences (introns) between individual coding segments (exons), as well as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. The terms "gene(s)" or "polynucleotide" or "nucleic acid" or "polynucleotide sequence(s)" are intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms arc intended to include one or more genes that map to a functional locus.
Also, the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host cell or can be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome. In addition to the plasmid form, a gene can, for example, be in the form of linear DNA or RNA. The term "gene" is also intended to refer to multiple copies of a particular gene, e.g., all of the DNA sequences in a cell encoding a particular gene product.
[0042] A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength.
Hybridization and washing conditions are well known and exemplified, e.g., in Sambrook, J., Fritsch, E. F.
and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (hereinafter ''Maniatis"). The conditions of temperature and ionic strength determine the "stringency" of the hybridization Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that

9 duplicate functional miry mes from closely related organisms. Post-in.bridization washes determine stringency' conditions. One set of conditions uses a series of washes starting As ith 6X
SSC. 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC. 0.5% SDS at 45 C for 30 min, and then repeated twice with 0.2X SSC.
0.5% SDS at 50 C for 10 min. For more stringent conditions, washes are performed at higher temperatures in which the washes are identical to those abo%e except for the temperature of the final two 10 nun %ashes in 0.2X
SSC. 0.5% SDS are increased to 60 C. Another set of highly stringent conditions uses two final washes in 0.IX
SSC. 0.1% SDS at 65 C. An additional set of highly: stringent conditions are defined by hybridization at 0.1 X
SSC. 0.1% SDS. 65 C and washed with 2X SSC. 0.1% SDS followed by 0.1 X SSC.
0.1"4 SDS
100431 Hybridization minims that the two nucleic acids contain complementary sequences. although depending on the stringency of the hybridization. mismatches between bases arc possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation. variables well known in the an. The greater thc degree of similarity or homology between two nucleotide sequences. the greater the value of Tin for hybrids of nucleic acids ha % ine those sequences, The relative stability (corresponding to higher Tin) of nucleic acid hybridizations decreases in the following order.
RNA:RNA. DNA:RNA. DNA:DNA. For hybrids of greater than 100 nucleotides in length. equations for calculating Tm have been denied (see. e.g.. Manialis at 9.50-9.51). For hybridizations with shorter nucleic acids, i.e.. oligonuclemides, the position of mismatches becomes more important, and the length of the oligonucleot ide determines its specificity (see. e.g.. Maniatis. at 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybriditable nucleic acid is at least about 15 nucleotides: more preferably at least about 20 nucleotides: and most preferably the length is at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as length of the probe.
100441 As used herein the term "codon-optimized" means that a nucleic acid coding region has been adapted for expression in the cells of ri given organism by replacing one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.
100451 The term "percent identity". as known in the art, is a relationship between two or more polypeptidc sequences or two or more poly nucleotide sequences, as determined by comparing the sequences, hi the art.
"identity" also means the degree of sequence relatedness between polypeptide or polynneleotide sequences. as the case can be. as determined by the twitch between strings of such sequences.
100461 As known in the art, "similarity." between tuo polypcptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the poly peptide to the sequence of a second polypepticie. Similarity can be between tno full sequences. or between a fragment of one sequence Mid a fragnmit of a second sequence whcria it the fragments are of comparable length or sin, or boneen a fragment of one sequence and the entirety of a second sequence.
100471 "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk., A. M. ed.) Oxford University Press. NY (1988):
Biocomputing: Informatics and Gem= Projects (Smith, D. W., ed.) Academic Press. NY (1993); Computer Analysis of Sequence Data. Part I (Griffin. A. M., and Griffin. H. (1. eds.) Humana Press. NJ (1994): Sequence Analysis in Molecular Biology (von Heinle. G.. ed.) Academic Press (1987): and Sequence Analysis Primer (Gribskov. M. and Devereux. J., eds.) Stockton Press. NY (1991). Preferred methods to determine identity arc designed to give the best match between the sequences tested. Methods to determine identity. and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using the Megalign pmgrain of the LASERGENE bioinfonnatics computing suite (DNASTAR Inc..
Madison. Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10.
GAP LENGTH PENALTY=I0). Default parameters for palmist., alignments using IC
ChMal method we're KTUPLE 1. GAP PENALTY=3. WINDOW=5 and DIAGONALS SAVED-5 1004111 Suitable nucleic acid sequences or fragments thereof (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% to about 75% identical to the amino acid sequences reported herein, at least about 80%. at least about 85% at least about 86 4. at least about 87%. at least about 88%. at last about 89%. or at least aboat 90% identical to the amino acid sequences reported herein, at least about 91%. at least about 92%. at least about 93%. at least about 94%. or at least about 95%
identical to the amino acid sequences reported herein, or at least about 96%. at least about 97%, at least about 98%. at least about 99% or about 100% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments are at least about 50%, at least about 55%. at least about 60%. uI least about 65%. at least about 70%. at least about 75%, at least about 76%. at least about 77%. at least about 78%. at least about 79%.
at least about 80%. at least about 81%. at least about 82%. at least about 83%, at least about 84%. at least about 85%. at least about 86%. at least about 87%. at least about 88%, at least about 89%. at least about 90%. at least about 91%. at least about 92%, at least about 93%. at least about 94%, at least about 9.5%. at least about 96%, at least about 97%, at least about 98%. at least about 99% or about 100% identical to the nucleic acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities/similarities but b pically ataxic a poly-peptide having at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, or at least 250 amino acids 100491 A DNA or RNA "coding region" is a DNA or RNA molecule which is transcribed and/or translated into a polypcptide in a cell in vitro or in vivo when placed under the control of appropriate regulatoiy sequences.
"Suitable regulatory regions" refer to nuckic acid regions located upstream (5' non-coding sequences). within, or downstream (3' non-coding sequences) of a coding region. and which influence the transcription. RNA
processing or stability, or translation of the associated coding region.
Regulatory regions include promoters.
translation leader sequences, RNA processing site. effector binding site and stem-loop structure. The boundaries of the coding region arc determined by a start codon at the 5' (amino) terminus and a translation stop eodon at the 3' (carboxyl) terminus A coding region can include, but is not limited to.
prokaryotic regions. cDNA from inRNA. genomic DNA molecules, synthetic DNA molecules. or RNA molecules. If the coding region is intended for expression in a ctikaryotic cell, a poly adcnylation signal and transcription termination sequence will usually be located 3(0 the coding region.
100501 An "isofonn" is a protein that has the same function as another protein but which is encoded by a different gene and can have small differences in its sequence 100511 A "paraloguc" is a protein encoded by a gene related by duplication within a gcnome.
100521 An "onhologue" is gene from a different species that has evolved from a common ancestral gene by spociation Normally. onhologues retain the same function in the course of evolution as the ancestral gene.
100531 "Open reading frame" is abbreviated ORF and means a length of nucleic acid, either DNA. cDNA or RNA. iltit comprises a translation start signal or initiation codon. such as an ATG or AUG. and a termination codon and can be potentially translated into a polypeptide sequence.
100541 "Promoter refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA. In general. a coding region is located S to a promoter.
Promoters can be isolated in their entirety from a native gene. or be composed of different demons derived from different promoters found in nature., or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types. or at different stages of development. or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that SilICL in most eases the exact boundaries of regulatory sequences lime not been completely defined. DNA fragments of different lengths can have identical promoter activity. A promoter is generally bounded at its 3' terminus b the transcription initiation sitc and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (cons eniently defined for example.
by mapping with nuclease 51). as well as protein binding domains (consensus sequences) responsible for the binding or RNA poly merase Several promoters are specifically identified by the present invention. however, one having ordinal) skill in the art would understand that an number of additional promoters capable of driving the expression in yeast would be included in the present invention.
100551 The term "linker" as used herein refers to a series of nucleotides or amine acids tliat connect 011C seelion of the chimeric polynticleotide or polypeptide to another section of the chimeric poly nucleotide of poly peptide.
In sonic embodiments. the linker serves a structural function.
10061 A coding region is "under the control" of transcriptional and translational control elements in a cell when RNA polymer:Ise transcribes the coding region into taRNA, which is then trans-RNA spliced (if the coding region contains introns) and translated into the protein encoded by the coding region.
100571 *Transcriptional and translational control regions" are DNA regulatoiy.
regions. such as promoters.
enhancers. terminators, and the like, that pros idc for the expression of a coding region in a host cell. In eukaryotic cells. poly adeny lation signals are control regions.
10058( As used herein the term "N-terminal region" refers to the portion of the amino acid sequence consisting of the most N-terminal amino acid residue up to the amino acid residue at position n/2. wherein n is the total number of residues in the sequence. As used herein the term "C-tenninal region" refers to the portion of the amino acid sequence consisting of the most C-terminal amino acid residue up to the amino acid residue at positionn/2. wherein a is the total number of residues in the sequence.
100591 The term "operably associated" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably associated with a coding region when it is capable of affecting the expression of' that coding region (i.e.. that the coding region is under the transcriptional control of the promoter). Coding regions can be operably associated to regulatory regions in sense or anti sense orientation.
100601 The term "expression." as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression can also refer to translation of niRNA into a poly peptide.

WO 2016/024215 PCT/1B2111511156 In 1 100611 The term "lignocellulosc" refers to material tlxit is comprised of lignin and cellulose. Examples of lignocx:Iluloses are prON hied herein and arc kilos, ii in the art. Examples of lignocellidosie materials include but are not limited to corn stover. straw. bagasse. switchgrass. paper, and wood.
100621 The "pentosc phosphate pathway" or "PPP" milers to a biochemical pathway that creates NADPH from gliicose.6-P. The PPP has both an oxidative phase and a non-oxidative phase.
There arc several cozy rues that have been identified to play a role in the PPP. including but not limited to glucose-6-P dehy drogenase.
gluconolactonase. 6-phosphogluconate dehydrogenase. ribulose-5-phosphate isomerase. ribose-5-phosplulic ketol-isomerase (RK 11). ribulose-5-phosphate 3-epirnentse (RPE I ), transketolase (TKL I ). and transaldolase (TALI).
10063) As used herein "xy.lose isomerase activity" refers to the ability or an enzyme to directly convert xylose to xyltilose. A isomerasc" or "XI" as used herein refers to a protein haying xylose isornerase activity. (EC
5.3.1.5) 100641 The term "chimeric" or "chimera" refers to a polynucleotide or poly peptide having a nucleotide or polypeptide sequence derived from two or more distinct parent sequences. A
"parent sequence" or "donor sequence" is a nucleotide or amino acid sequence used as a source sequence to mite the chimeric polynticleotide or poly peptide.
100651 As used herein the term "XYMI" or "XY1x12" refers to a xylose isomerase coding sequence or poly-peptide isolated from an uncultured bacteritini as described by Parachin and Gonva-Grauslund. "Isolation of xylose isomerasc by sequence- and function-based screening from a soil metagcnome library," Dimechnology Biofuels 4(1) 9 (2(111).
100661 As used herein, the term "anaerobic" refers to an organism. biochemical reaction, or process that is active or occurs under conditions of an absence of gaseous 02.
100671 "Anaerobic conditions" are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to usc it as a (C111141;11 electron acceptor. Anaerobic conditions can be achieved by sparging a fermentation medium (vith an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor.
Alternatisely. anaerobic conditions can be achieved by the microorganism consuming the available oxygen of fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor.
100681 "Aerobic metabolism" refers to a biochemical process in which oxy gen is used as a terminal electron acceptor to convert energy. typically in the form of ATP. from carbohydrates.
Aerobic metabolism typically occurs, for example, via the electron transport chain in mitochondria in eukaryotes, wherein a single glucose molecule is metabolized completely. into emboli dioxide in the presence of oxygen.
100691 In contrast. "anaerobic metabolism" refers to a biochemical process in which oxygen is not the final acceptor of electrons generated. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than osygen serve as the terminal electron acceptor, and substrate level phosphorylation. in which no exogenous electron acceptor is used and products of an intermediate oxidation state am generated via a "fermentative pathway."
100701 In "fermentative pathways", the amount of NAD(P)H generated bs glycolysis is balanced by the consumption of the same amount of NAD(P)H in subsequent steps. For example, in one of the fennentative pathways of certain yeast strains. NAD(P)H generated through glycolysis donates its electrons to acetaldehyde.

WO 2016/024215 PC1./11320151056101 yielding ethanol. Fermentative pathways are usually active under anaerobic conditions but can also occur under aerobic conditions, under conditions where NADH is not fully oxidi/ed via the icspiratoiy chain.
100711 As used herein, the term "end-product" refers to a chemical compound drit is not or cannot be used by a cell, and so is excreted or allowed to diffuse into the extmcelltilar environment. Common examples of end -products from anaerobic fermentation include. but are not limited to. ethanol, acetic acid. formic acid, lactic acid.
hydrogen. and carbon dioxide.
100721 As used herein, *cofactors" arc compounds involved in biochemical reactions that are recycled within the cells and remain at approximately steady slate levels. Common examples of cofactors involved in anaerobic fermentation include. but arc not limited to. NAD+ and NADP+. In metabolism, a cofactor can act in oxidation-reduction reactions to accept or donate electrons. When organic compounds are broken down by oxidation in metabolism, their energy can be transferred to NAD+ by its reduction to NADH.
to NADP+ by its reduction to NADPH. or to another cofactor. FAD+. by its reduction to FADI12. The reduced cofactors can then be used as a substrate for a reductase.
100731 As used herein. a "pathway' is a group of biochemical reactions that together can convert one compound into another compound in a step-wise process. A product of the first step in a pathway can be substrate for the second step. and a product of the second step can be a substrate for the third, and so on.
Pathways of the present invention include, but are not limited to. the pentose phosphate pathway. the xylosc utilization pathway. the ethanol production pathway, and the gly cerol production pathway. The term "recombination" or "recombinant" refers to the physical exclumge of DNA
between mo identical (homologous), or nearly identical. DNA molecules. Recombination can be used for targeted gene deletion or to modify the sequence of a gene. The terms "recombinant microorganism" and "recombinant host cell" arc used interchangeably herein and refer to microorganisms that tune been genetically modified to express or over-express endogenous poly nucleotides. or to express heterologou.s polynucleotides. such as those included in a vector, or which have a modification in expression of au endogenous gene.
100741 By "expression modification" it is meant that the expression of the gene. or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptidc subunits, or activity of one or more poly peptides or polypeptide subunits is up regulated or down-regulated.
such that expression, level, or activity, is greater than or less than that observed in the absence of the modification.
100751 The term "iron metabolism" refers to the process by which a cell regulates the intracellular level of iron.
The term "protein associated with iron metabolism" refers to a protein involved in the regulation of intracellular hon. including, e.g.. a protein that imports, exports, binds, and/or sequesters iron or a protein that controls the expression of a gene that encodes for a protein that impons. exports. binds, and/or sequesters iron. The term "Fe/S cluster biosynthesis" refers to the biosynthesis of Fe/S clusters, including. e.g.. the assembly and loading of Fe/S clusters. The term "Fe/S cluster biosynthesis genes". "Fe/S cluster biosynthesis proteins" or "Fe/S cluster biosynthesis pathway" refers to those polynucleotides and/or genes that arc involved in the biosynthesis of Fe/S
clusters, including. e.g.. the assembly and loading of Fe/S clusters.
I00761 In one aspect of the invention, genes or particular polynucicotide sequences are panially. substantially, or completely deleted. silenced, inactivated, or down-regulated in order to inactivate the cozy matte activity they encode. Complete deletions provide maximum stability. because there is no opponunity for a reverse mutation to restore function. Alternatively, genes Can be partially. substantially, or completely deleted, silenced. inactivated.

or down-regulated by insertion. deletion. removal. or substitution of nucleic acid sequences that disrupt the function and/or expression of the gene.
Xylose Isomerast Polypepticles 10077) Thc present invention provides host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism and (b) at least one gene encoding a polypeptide having xylose isomerase activity the IISC thereof. In some embodiments. the host cell hetcroloaousts expresses the polypeptide. In some embodiments. the licterologously expressed poly:peptide is a naturally occurriug polypeptidc. In some cmbodinterds. the heterologously expressed polypeptide is recombinant. In some embodiments, the beterologously expressed polypeptidc is a chimeric polypeptide.
100781 In some embodiments, the polypeptide has an amino acid sequence having at least 80%. at least 155%. at least 86%. at least 87%, at least 88%. at least 89%. at least 90%. at least 91%, at least 92%, at least 93%. at last 94%. at least 95%. at least 96%. at least 97%. at least 98%. or at least 99%
sequence identity with any one of SEQ ID NOs: I. 3, 5. 7. 9, II. 13. 15. 17. 19. 21. 23. 25. and/or 27. In some embodiments, the poly-peptide has an amino acid sequence of SEQ ID NOs: 1.3, 5.7.9. I I. 13. 15, 17, 19,21. 23.
25. 01 27 In some embodiments.
the polypeptide is encoded by a polynueleottde sequence having at least 50%.
at least 55%. at least 60%, at least 65%. at least 70%. at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at lyrist 80%, at least 81%.
at least 82%. at least 83%. at least 84%. at least 85%. at least 86%. at least 87%. at least 88%, at least 89%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%. at least 95%. at least 96%, at least 97%. at least 98%. oi at least 99"'a sequence identity %%itlt any our of SEQ 1D NOs. 2, 4.
6, 8. 10, 12. 14, 16. 18, 20, 22. 24. 26.
and/or 28. In some embodiments, the polypeptide is encoded by a polynucleotide sequence of SEQ ID NOs: 2,4, 6. 8, IQ 12. 14, 16. IS. 20. 22. 24. 26, or 28. lit sonic embodiments, the polypeptide has an ainino acid sequence having at least 80%. at least 85%. at least 86%, at least 87%. at least 88')).
at least 890/.. at least 90% at least 910.. at least 92%. at least 93%. at least 94%. at least 95%, at least 90%. at least 97%. at least 98%, or at least 99 A, sequence identity with am one of SEQ ID NOs: I. 3, 5.7. 9, 11. 13, 15, 17, 19, 21, 23, 25, 27, 35. 37. 39.
and/or 41. In some embodiments, the Fmk) peptide has an amino acid sequence of SEQ ID NOs: , 3. 5, 7, 9. 11.
13. 15. 17, 19. 21, 23, 25, 27, 35. 37, 39, or 41. In some embodiments, the polypeptidc is encoded by a poly nucleotide sequence having at least 50%. at least 55%. at least 60%, at least 65%, at least 70%. at least 75%, at least 76%. at least 77%. at least 78%, at least 79%. at least 80% at least 81%. at least 82%. at least 133%. at least 84%, at least 85%, at least 86%, at least 87%, at least 88%. at !cast 89 /0, at least 90*/a, at least 91%. at least 92%. at least 93%. at least 94%, at least 95%, at least 96%. at least 97%. at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 2. 4.6. 8. 10. 12. 14. 16. 18. 20. 22.
24.26, 28. 36, 38. 40, and/or 42 . In some embodiments. the polypeptide is encoded by: a polynticlemide sequence of SEQ ID NOs: 2. 4.6.8. 10. 12, 14, 16.18. 20. 22. 24. 26,28,36, 38.40. or 42.
100791 In some embodiments. the pots:peptide has an amino acid sequence having at least 150%. at least 85%, at least 86%. at least 87%. at least 88% at least 89%. at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%. at !cast 96%. at least 97%. at least Ott%.. oral least 99%
sequence identity with the amino acid sequence of SEQ ID NO. I. In some embodiments. the polypeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: I.
100801 In some embodiments. the polypeptide has an amino acid sequence having at least 80%, at least 85%. at least 86%, at least 87% at least 88%. at least 89%. at least 90%. at least 919'0. at least 92%. at least 93%. at least 944i. at Cast 95%. at le.ast 90%. at least 97% at kast 98%. oral least 99%
sequence identits with the amino acid sequence of SEQ ID NO: 3. hi some embodiments, the polypeptide has an amino acid sequence having 100", sequence identity with the amino acid sequence of SEQ ID NO: 3.
100811 In sonic embodiments. the polypeptidc has an amino acid sequence having at least 80%. at least 85%. at least 86%. at least 87%, at least 88% at least 89%. at least 90%. at least 91%. at kast 92%, at least 93%. at least 94%, at least 95%. at least 96%. at least 97%, at least 98%, or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the polypeptide has an amino acid sequence having 100% sequence 'details with the amino acid sequence of SEQ NO: 5.
100821 In some embodiments. the polypeptide has an amino acid sequence having at least 80%. at least 85%. at least 86%. at least 87%. at least 88% at least 89%. at least 90%. at least 91%, at least 92%. at least 93% at least 94%. at least 93%, at least 96%. at least 97% at least 98%. or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the polypeptide has an amino acid sequence having WO%
sequence identity with the amino acid sequence of SEQ ID NO: 7.
100831 In some embodiments, the polspeptide has an amino acid sequence having at least 80% at least 85%. at least 80%, at least 87%. at least 8(5 ,0, at least 89 ,0, at least 90%, at least 91%. at least 92%, at least 93%. on least 94%, at least 95% at least 96%, at least 97%, at least 98%. or at least 99%
sequence identity with the amino acid sequence of SEQ 1D NO: 9. In some embodiments. the polypeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: 9.
1008.11 In sonic embodincins. the pokspeptide has an amino acid sequence liming at least 80%, at least 85% at least 86%. at least 87%. at least 88%, at least 89%. at least 90% at least 91%, at least 92%, at least 93%. at least 94%, at least 95% at least 96%. at least 97%. at least 98% or at least 99%
sequence identity with the amino acid segue= of SEQ ID NO: II. In some embodiments, the polypeptide has an amino add sequence having 100%
sequence identits µµith the amino acid sequence of SEQ ID NO: 11.
10085) In some embodiments, the polypeptide has an amino acid sequence leaving at least 80%. at least 85% at least 86%, at least 87%, at least 88%. at least 89%. at least 90%. at least 91%. at least 92%. at least 93%, at least 94%, at least 95% at least 96%. at least 97%. at least 98%. or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the polypeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: It.
100861 In sonic embodiments, the potypeptide has an amino acid sequence has*
at least 80%, at least 85%, at least 80%, at least 87%, at least 88%. at least 89%, at least 90%. at least 91%. at least 92%. at least 93%. at least 94%, at least 95%, at least 96%. at least 97%, at least 98%. or at least 99%
sequence identity nith ilk amino acid sequence of SEQ ID NO: 13. In some ernboditnents, the polypeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: 15.
100871 In sonic embodiments. the polypeptide has an amino acid sequence having at least 80%. at least 85%, at least 86%, at least 87%. it least 88%, at least 89% at least 90%. at least 91%. at least 92%, at least 93%. at least 94%, at least 95%. at !Cast 96%. at least 97%. at least 98%, or at least 99%
sequence identits with the amino acid sequence of SEQ ID NO: 17. In some embodiments, the polspeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: 17.
100881 In some embodiments. the polypeptide has an amino acid sequence having at least 80%. at least 85%, at least 150%, at least 87%. at least 88%. at least 89%. at least 90%, at least 91%. at least 92%. at least 93%. at least =

94% at least 95%, at least 96%. at least 97% at least 98%, or at least 99%
sequence identity ss ith the amilk, acid sequence of SEQ ID NO: 19. In some embodiments, the polypeptide has an amino acid sequence having tixt'!1, sequence identity with the amino acid sequence of SEQ ID NO: 19.
100891 In some embodiments, the polypeptide has an amino acid sequence having at least 80%. at least 85%. at least 86%. at least X7%, at least 88%. at least 89%, at least 90%. at least 91%. at least 92%, at least 93%. at least 94%. at least 95%, at least 96%, at least 97% at least 98%. or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments. the polypeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO:21 .
100901 In sonic embodiments. the polypeptide has an amino acid sequence tuning at least 80%, at least 8.5%. at least 86%, at least 87% at least 88%. at least 89%. at least 90%. at least 91%
at least 92%. at least 93%. at least 94%. at least 95%. at least 96%. at least 97% at least 98%, or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 23. In some embodiments. the polypeptide has an amino acid sequence basing 100%
sequence identity. with the amino acid sequence of SEQ ID NO: 23.
100911 In some embodiments, the polypepncle has an amino acid sequence having at least 80%, at least 85%. at least 86%. at least 87%. at least 88%. at least 89% at least 90%. at least 91%. at least 92%. at least 93% at least 94%. at least 95%, at least 96%. at least 97% at least 98%. or at least 99%
sequence Went ity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the polypeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: 25.
100921 In some embodiments, the poly peptide has an amino acid sequence hay ing at least 80r,0, at least 85%. at least 86%, at least 87%, at least 88%, at least 89%. at least 90%. at least 91%, at least 92%, at least 93%. at least 94%. at least 93%. at least 96%. at Was( 97% at least 98%, or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 27. In some embodiments, the pohlieptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: 27.
100931 In some embodiments, the polypeptide has an amino acid sequence having at least 80%. at least 35%. at least 86%, at least 8'7% at least XX%, at least 89% at least 90%. at least 91%. at least 92%, at least 93%. at least 94% at least 95%, at least 96%, at least 97%. at least 98%, or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 33. In some embodiments. the polypeptide has an amino acid sequence having 100%
sequence identity with the amino acid sequence of SEQ ID NO: 35.
100941 In some embodiments, the polypeptide has an amino acid sequence having at least 80%. at least 35%. at least 86%, at least 87%, at least 88%. at least 89%, at least 90%, at least 91%. at least 92%. at least 93%. at least 94%, at least 95%. at least 96%. at least 97%, at least 98%. or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 37. In some embodiments. the poly peptide has an amino acid sequence !wing 100%
sequence identity with the amino acid sequence of SEQ ID NO: 37.
100951 In some embodiments, the polypeptide has an amino acid sequence having at least 80%. at least 85%. at least 86%, at least 87%, at !cast 88%, at least 89% at least 90%. at least 91%
at least 92%, at least 93%, at least 94%, at least 95%. at least 96%. at least 97%. at least 98%, or at least 99%
sequence identity with the amino acid sequence of SEQ ID NO: 39. In some embodiments. the polypeptide has an amino acid sCqucicc having 100%
sequence identity with the amino acid sequence of SEQ ID NO: 39.
100961 In some embodiments, the polypeptide has an amino acid sequence having at least 80%. at least 85%. at least 86%, at least 87% at least 88%. at least 89%, at least 90%. at least 91%. at least 92%. at least 93%, at least = CA 02957707 2017-02-03 94%. at least 95%. at least 96%. at least 97% at least 98%. oral least 99%
sequence identiiy. uith the amino acid sequence of SEQ ED NO: 41. In some embodiments, the polypeptide has an amino acid sequence having t00%
sequence identity with the amino acid sequence of SEQ ID NO: 41.
100971 In some embodiments, the polypeptide is encoded by a polynneleotide sequence having at least 50%. at least 53%, at least 60%. at least 65%. at least 70%. at least 73%. at least 76%. at least 77%. at least 78%, at least 79%. at least 80%. at least 81%, at least 82%. at least 83% at least 84%. at least 85%. at least 86% at least 87%
at least 88%. at least 89% at least 90%. at least 91%. at least 92%, at least 93%. at least 94%. at least 95%. at least 96%. at least 97%, at least 98%, or at least 99%soquence identity with the nucleotide sequence of SEQ ID
NO: 2. In some embodiments, the polypeptide is encoded by a polynneleotide sequence of SEQ 1D NO: 2.
100981 In some embodiments, the polypcptide is encoded by a polynucleotide sequence having at least 50%. at least 55%. at least 60%. at least 65%. at least 70% at least 75%, at least 76%. at least 77% at least 78%. at least 79%, at least 80%. at least 81%, at least 82% at least 83% at least 84%. at least 85%, at least 86% at least 87%.
at least E18%. at least 89% at least 90%. at least 91%. at least 92%. at least 93%. at least 94%. at lost 95%. at least 96%, at least 97%, at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 4.
100991 In some embodiments, the polypeptide is encoded by a polynueleotide sequence having at least 50% at least 55%. at least 60%, at least 65%, at lost 70%. at least 75% at least 76%.
at least 77%, at least 78% at least 79%, at least 80%. at least 81%. at least 82% at least 83%. at least 84%, at least 35%, at least 86%, at lost 87%, at least 88%. at least 89%. at least 90%, at least 91%. at loam 92%, at least 93% at least 94%, at least 93%, at least 96%. at lost 97%, at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 6. In some embodiments, the polypeptide is encoded by a polynucleotide sequence of SEQ ID NO: 6.
1001001 In some embodiments, the poly-peptide is encoded by a polymieleotide sequence lining at least 50% at least 55%. at least 60%, at least 65%, at least 70%, at least 75%. at least 76%. at least 77%, at least 78%, at least 79%. at least 80%. at least 81%. at least 82% at least 83%. at least 84%. at least 85%. at least 86%. at least 87%
at least 88%, at least 89%. at least 90%. at least 91%, at least 92%. at least 93%, at least 94%. at least 95%, at lost 96%, at least 97%, at least 98%, or at lost 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 8. In some embodiments, the poly peptide is encoded by a poly nucleotide sequence of SEQ ID NO: 8.
1001011 In some embodiments, the polypeptide is encoded by a polynucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at lost 78%, at least 79%, at least 110%. at least 81%. at least 82%. at least 83%. at least 84%. at least 85%, at least 86% at least 87%, at least 88%. at least 89%, at least 90%. at least 91%, at least 92%. at least 93%. at least 94% at least 95%. at least 96%. at least 97%= at least 98%. oral least 99% sequence identity with the nucleotide sequence of SEQ ED
NO: 10.1n some embodiments, the poly-peptide is encoded by a polynneleolide sequence of SEQ ID NO: 10, 1001021 In some embodiments. the potypeptide is encoded by a polynueleotide sequence having at least 50%. at least 55%. at least 60%. at least 65%, at least 70%. at least 75%. at least 76%. at least 77%. at least 78%, at least 79%. at least 80%. at least 81%. at least 82%. at least 83%. at least 84% at least 85% at least 86%, at least 87%
at least 88%. at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at lost 97%, at least 98%, oral least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 12. In some embodiments. the polypeptidc is encoded by a poly.micleotide sequence of SEQ ID NO: 12.
1001031 In some embodiments. the poly peptide is cncodcd by a polynuelcolide sequence having at least 50%. at = CA 02957707 2017-02-03 least 55%. at least 60%. at least 65% at least 70%, at least 75%, at least 76%, at least 77%. at least 78%, at least 79%, at least 80%. at least 81%. at least 82% at least 83%, at least 841-6, at least 85%. at least 86%, at least 87%.
at least 88%. at least 89% at least 90%. at least 91%, at least 92%, at least 93%, at least 94%. at least 95%. at least 96%. at least 97% at least 93%, or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 14. In some embodiments. the p0 peptide is encoded by a polynucleotide sequence of SEQ ID NO: 14.
1001041 In some embodiments. the polypeptide is encoded by a polynucleotide sequence having at !mast 50% at least 55%. at least 60%, at least 65%, at least 70%. at least 75%. at least 76%. at least 77%. at least 78%, at least 79%, at least 80%. at least 81%. at least 82% at least 83%, at least 84%, at least 85% at least 86% at least 87%.
at least 88%. at least 89% at least 90%, at least 91%. at least 92%. at least 93%. at least 94%. at least 95%. at least 96%. at least 97%. at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 16. In some embodiments, the polypeptide is encoded by a polynuelcotide sequence of SEQ ID NO: 16.
1001051 In some embodiments, the polypeptide is encoded by a polynueleotide sequence having at least 50%. at least 55%, at least 60%, at least 65%. at least 70%, at least 75%. at least 76%. at least 77%, at least 78%. at least 79%, at least 80%. at least 81%. at least 82% at least 83% at least 84%, at least 85%. at least 86%, at least 87%.
at least 88%. at least 89% at least 90%. at least 91%. at least 92%. at least 93%. at least 94%. at least 95% at least 96%. at least 97%. at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: IS. In some embodiments, the polypeptide is encoded by a polynucleotidc sequence of SEQ ID NO: 18.
1001061 In some embodiments, the poly-peptide is encoded by a polynucleotide sequence having at least 50% at least 55%. at least 60%, at least 65%. at least 70%, at least 75%. at least 76%. at least 77%, at least 78%, at least 79%, at least 80%. at least 81%. at least 32%. at least 83%. at least 84%, at least 85%. at least 86%, at least 87%
at least 88%, at least 89%, at least 90%, at least 91%. at least 92%. at least 93%. at least 94% at least 95%, at least 96% at least 97%. at least 98% oral least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 20. In some embodiments, the poly peptide is encoded by a polynucleotide sequence of SEQ ID NO: 20, 1001071 In sonic embodiments. the polypeptide is encoded by a poly nucleotide sequence having at least 50%. at least 55%, at least 60%, at least 65%, at least 70%. at least 75%. at least 76%. at least 77%, at least 78%, at least 79%, at least 80%. at least 81%. at least 82%. at least 83%, at least 84%, at least 85%. at least 86%, at least 87%, at least 88%, at least 89%. at least 90%. at least 91%. at least 92%, at least 93%. at least 94%. at least 95%. at least 96%. at least 97%, at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 22. In sonic embodiments, the polypcptidc is encoded by a polyinicleotidc sequence of SEQ ID NO: 22.
1001081 In some embodiments, the polypeptidc is encoded by a polynueleotide sequence having at least 50%, at least 55%. at least 60%. at least 65%, at least '70%. at least 75%. at least 76%. at least 77%. at least 78%. at least 79%, at least 80%. at least 81% at least 82,4, at least 83% at least 84%. at least 85%, at least 86%, at least 87%
at least 880/a, at least 89%, at least 90%. at least 91%. at least 92%. at least 93%, at least 94%, at least 9504 at least 96%. at least 97%. at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 24. In sonic embodiments, the polypeptide is encoded by a polynucicotide sequence of SEQ ID NO: 24.
1001091 In some embodiments. the poly-peptide is encoded by a polynuclentide sequence having at least 30%. at least 55%, at least 00%, at least 65%. at least 70%, at least 75%, at least 76%, at least 77%. at least 78%, at least 79%, at least 80%. at least 81%, at least 82%. at least 83%. at least 84 A,.
at least 85%. at least 86%, at least 87%.
at least 88%, at least 89%, at least 90%. at least 91%, at least 92%. at least 93%, at least 94% at least 95%. at least 96%. at least 97%. at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID

WO 1(116/924215 PCT/1132015/056101 NO: 26. In some embodiments, the polypeptide is encoded by a polynticleotide sequence of SEQ ID NO: 26.
1001101 In some embodiments, the poly peptide is encoded by a pot) nucleotide sequence having at least 50% at least 53%. at least 60% at least 65% at least 70%. at least 75% at least 76%.
at least 77%, at least 78%, at least 79%. at least 80%, at least 81%. at least 82% at least 83%. at least 84%, at least 85% at least 86%. at least 87%, at least 88%. at least 89%. at least 90%. at least 91%, at least 92%, at least 03%, at least 94%. at least 95%. at least 96%. at least 97%. at least 914%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 28.1n some embodiments, the poly-peptide is encoded by a polymicleotide sequence of SEQ ID NO: 28.
1001111 ht some embodiments, the poly-peptide is encoded by a polynucleotide sequence having at least 50% at least 55%. at least 60%. at least 65%. at least 70%. at least 75%, at least 76% at least 77%. al least 78%, at !cast 79%, at least 80%. at least 81%. at least 82%. at least 83%. at least 84%, at least 85% at least 86%. at least 87%.
at least 88%, at least 89%. at least 90%, at least 91%. at least 92% at least 93%. at least 94%. at least 95% at least 96%. at least 97%, at least 98% or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 36.1n some embodiments, the polypeptide is encoded by a polytmeleolide sequence of SEQ ID NO: 36.
1001121 In some embodiments. the polypeptide is encoded by a poly -nucleotide sequence having at least 50%, at least 55%. at least 60%. at least 65%, at least 70%, at least 75%. at least 76%. at least 77%. at least 78%, at least 79%, at least 80%, at least 81%. at least 82%, at least 83%, at least 84%, at least 85% at least 86%. at least 87%
at least 88%, at least 89% at least 90%. at least 91%. at least 92%. at least 93%. at least 94%. at least 95%. at least 96%. at least 97%, at least 98% or at least 99% sequence identity. with the nucleotide sequence of SEQ ID
NO: 38. In sonic embodiments, the poly:peptide is encoded by a polvnucleotide sequence of SEQ ID NO: 38.
1001131 In sonic embodiments, the poly-peptide is encoded by a poly nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%. at least 70%, at least 75%. at least 76%, at least 77% at least 78%. at least 79%. at least 80%. at least 81%. at least 82% at least 83%, at least 114%. at least 85% at least 86%. at least 87%.
at least Int%, at least 89%, at least 90%. at least 91%, at least 92%. at least 93%, at least 94%, at cast 95%. at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 40.1n some embodiments. the polypeptide is encoded by a polynucleotide sequence of SEQ ID NO: 40.
1001141 In sonic embodiments, the poly-peptide is encoded by a polynueleotide sequence having at least 50%, at least 55%. at least 60%. at least 65%. at least 70% ru least 75%, at least 76%
at least 77%. at least 78%, at least 79%, at least 80%. at least 81%. at least 82% at least 83%. at least 84%. at least 85% at least 86%, at least 87%, at least 88%, at least 89% at least 90%, at least 91%, at least 92%, at least 93% at least 94%. at least 95%. at least 96%. at least 97%, at least 98%. or at least 99% sequence identity with the nucleotide sequence of SEQ ID
NO: 42. In sonic embodiments, the poly peptide is encoded by a polynucleotide sequence of SEQ ID NO: 42.
I001119 The present invention involves the heterologous expression of one or more polypeptides having xylose isomerase activity. It is understood by one of ordinary. skill in the art that any polypeptide having sylose isomerasc activity or any polynucicotide encoding such a polypeptide may be used according to the present invention. Accordingly, this invention is not limited to the list of example xylose isomerase polypeptides provided. It is understood that nucleotide sequences encoding an) of the pol)peptidcs defined aboAc arc expressly included in the present invention. Further, any nucleotide sequence that comprises one or more amino acid substitutions, insertions and/or deletions, but that are within the ranges of identity or similarity as defined herein arc exTutssly included in the invention. However, the polypeptidcs having xylose isomerasc activity share certain conserved motifs In one embodiment, the nucleotide sequence of the invention encodes a xy lose , isomerase amino acid sequence comprising a xylose isomerase signature sequence as defined, e.g., by Meaden et al. (1994, Gene, 141 : 97-101): VXW[GP]GREG[YSTA]
(present at positions 188-196, relative to SEQ ID NO: 11) and [LIVMJEPKPX[EQW
(present at positions 233-240, relative to SEQ ID NO: 11), wherein "X" can be any amino acid and wherein amino acids in brackets indicates that one of the bracketed amino acids can be present at that position in the signature sequence. A xylose isomerase amino acid sequence of the invention can further comprise the conserved amino acid residues His-103, Asp-106, and Asp-341, which constitute a triad directly involved in catalysis, Lys-236 plays a structural as well as a functional catalytic role, and Glu-234 (relative to SEQ
ID NO: 11), which is involved in magnesium binding (Vangrysperre et al., "Localization of the essential histidine and carboxylate group in D-xylose isomerases,"
Biochem. J.
265: 699-705(1990); Henrick et al., "Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A
resolution, respectively," J. Mol. Biol. 208: 129-157 (1989); Bhosale et al., "Molecular and industrial aspects of glucose isomerase," Microbiol. Rev. 60: 280-300 (1996)).
Amino acid positions of the above signature sequences and conserved residues refer to positions in the reference amino acid sequence of the B. thetaiotaomicron xylose isomerase of SEQ ID NO: 11. In amino acid sequences of the invention other than SEQ
ID NO: 11, the amino acid positions of one or more of the above signature sequences and conserved residues are present in amino acid positions corresponding to the positions of the signature sequences and conserved residues in SEQ ID NO: 11, for example in a ClustalW (1.83 or 1.81) sequence alignment using default settings. The skilled person will know how to identify corresponding amino acid positions in xylose isomerase amino acid sequences other than SEQ ID NO: 11 using amino acid sequence alignment algorithms as defined hereinabove. These regions and positions will tolerate no or only conservative amino acid substitutions. One having ordinary skill in the art would understand that even conserved motifs can remain functional with conservative amino acid substitutions, and such substitutions are envisioned by the present invention. Amino acid substitutions outside of these regions and positions are unlikely to greatly affect xylose isomerase activity.

20a [00116] Additional structural features common to XIs have been described, e.g., by Chang et al., "Crystal Structures of Thermostable Xylose Isomerases from Thermus caldophilus and Thermus thermophiles: Possible Structural Determinants of Thermostability," J. Mol. Biol. 288:623-34 (1999), and RCSB Protein Data Bank, "Xylose Isomerase From Thermotoga neapolitana,"
http://wwvv.rcsb.org/pdb/explore/explore.do?structureId=1A0E, last accessed June 29, 2014, at 5:15pm. There are several known metal binding sits in the XI
sequence, including at residues Glu-234, Glu-270, His-273, Asp-298, Asp-309, Asp-311, and Asp-341. One having ordinary skill in the art would understand that any deletions or non-conservative substitutions at any one or more of these residues may lead to a decreased functionability of the resulting XI.
[00117] In some embodiments, a host cell is engineered to express one or more of the xylose isomerase polypeptides. In some embodiments, the host cell is a fungal cell, e.g. a yeast cell, e.g. a S. cerevisiae cell. In some embodiments the host cell is modified to have mutations affecting at least one gene encoding a protein of the pentose phosphate pathway. In some embodiments, the host cell has at least one mutation affecting the expression of at least one of XKS1, RKI1, RPE1, TKL1, TALI, or a combination thereof In some embodiments, the host cell has one or more mutations that correlate with an increase in the expression or an up-regulation of one or more of XKS1, RKI1, RPE1, TKL1, and/or TALI. In some embodiments the host cell can be modified through the heterologous expression of one or more polynucleotides encoding XKS I, RKI1, RPE1, TKL I, and/or TALL In some embodiments, the host cell has one or more mutations that correlate with a decrease in the expression or down-regulation of one or more of XKS1, RKI1, RPE1, TKL1, and/or TALL In some embodiments, the host cell has a modification of an endogenous aldose reductase. In some embodiments, the aldose reductase is GRE3. In some embodiments, the host cell has a deletion or disruption of all or part of the endogenous GRE3 gene. In some embodiments, the aldose reductase gene is YPR1. In some embodiments, the host cell has a deletion or disruption of all or part of the endogenous YPR1 gene. In some embodiments, the host cell has a deletion or disruption of all or part of both the endogenous GRE3 gene and the endogenous YPRI gene. In some embodiments, the host cell has a modification of PGM I and/or PGM2. In some embodiments, the host cell overexpresses PGM1 and/or PGM2. In some embodiments, the host cell has increased levels of Pgml and/or Pgm2 polypeptide and/or mRNA relative to a comparable host cell lacking a modification of PGM I and/or PGM2. In some embodiments, the host cell is a modified industrial yeast strain.
1001181 In some embodiments, the host cell comprises a deletion or disruption of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis as described in, e.g., U.S. Patent Application Publication No. 2014/0186930. In some embodiments, the host cell produces less glycerol than a control recombinant microorganism without deletion or disruption of said one or more endogenous enzymes that function to produce glycerol and/or regulate glycerol synthesis. In some embodiments, the one or more endogenous enzymes that function to produce glycerol are encoded by a GPD1 polynucleotide, a GPD2 polynucleotide, or both a GPD1 polynucleotide and a GPD2 polynucleotide. In some embodiments, one or both of the endogenous GPD1 and/or GPD2 genes are modified by mutation or deletion. In some embodiments, the host cell comprises a heterologous ADHE sequence. In some embodiments, the heterologous ADHE is from Bifidobacterium adolescentis. In some embodiments the native STL1 gene is upregulated by either modifying the promoter of the native copies or by introducing additional copies of STL1. In some embodiments the host cell comprises an ortholog of the native STL 1. In some embodiments the native ACS2 gene is upregulated by either modifying the promoter of the native copies or by introducing additional copies of ACS2. In some embodiments the host cell comprises an ortholog of the native ACS2 or ACS1 gene.
1001191 In some embodiments, the host cell comprises one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism. In some embodiments, the host cell comprises one or more mutations in one or more endogenous genes encoding an iron uptake protein, iron utilization protein, and/or an iron/sulfur (Fe/S) cluster biosynthesis protein. In some embodiments, the host cell comprises one or more mutations in one or more endogenous genes encoding a polypcptide affecting iron metabolism or Fe/S cluster biosynthesis. In some embodiments, the host cell is a recombinant yeast cell. In some embodiments, the recombinant yeast cell comprises one or more mutations in one or more of an endogenous gene selected from the group ISUL Y1111, NFSI AFT1, AFT2, YAPS, FRA1, FRA2. GREX3, GREX4. CCC1, and combinations thereof. In some embodiments, the recombinant yeast cell comprises one or more mutations in one or more of an endogenous gene which is homologous to one or more of an S. cerevisiae gene selected from the group ISUl, YFH1, NFSI, AFT], AFT2, YAPS, FRA1, FRA2, GREX3, and GREX4. and CCC1. In some embodiments, the recombinant yeast cell comprises a mutation in the endogenous AFT1 gene that results in iron-independent activation of the iron regulon such as the AFT1-1up or AFT2-1up alleles (Rutherford et al., 2003). In some embodiments, the recombinant yeast cell comprises a deletion or disruption of YAPS and/or CCC I and/or a nintat ion in the endogenous AFT1 or AFT2 gene that results in iron-independent activation of the iron regulon such as the AFT - 1 up or AFT2- I up Melts. In some embodiments, the host cell comprises one or more =mins in one or more endogenous genes selected from FRAL FRA2. GREX3. and GREX4. wherein the one or more imitations results in increased Aftl and/or Aft2 activity. In sonic embodiments, the increased Aft I
and/or MU activity results in the increased expression of Aril and/or Ara target genes. In some embodiments.
the one or more mutations in AFT I. AFT2. FRA I. FRA2. GREX3. and/or GREX4 prevent or limit AFT! and/or AFT2 from forming a complex with Grx3. Gr.x4. Fral. and/or Fm2.
1001201 In sonic embodiments, the host cell expresses one or more heterologous genes encoding a protein that is associated with iron metabolism. in some embodiments, the heterologous gene confers 0111 the recombinant yeast cell an increased ability to utilize xylosc as compered to a similar yeast cell lacking the heterologous gene. In sonic embodiments. the heterologous gene is AFT1. AFT2, and/or an onhologue thereof. In some embodiments.
the heterologous gene encodes a polypeptide having iron transpon activity. In sonic embodiments, the heterologous gene encode a protein that increases the activity and/or expression of A111 and/or Aft2 In some embodinvnts. the heterologous gene is a target of Aft I and/or Aft2. In some embodiments, the heterologous gene is constitutively expressed. In some embodiments. the hetcrologous gm is overexpressed. In some embodiments. the heterologous gene encodes a protein that suppresses a gene or protein that suppresses All 1 and/or Af12 activity and/or expression. In some embodiments, the heterologous gene encodes a protein that suppresses a gene or protein that suppresses the activity and/or expression of one or more don usticam targets of Aft I and/or Al12.
1001211 In some embodiments, the host cell comprises one or more imitations in the endogenous 1SU 1 gene that results in a poly peptide comprising at least one amino acid substitution selected from the group consisting of D71N. D71G. and S9IiF. wherein the position of the substitution is relative to the amino acid positions of SEQ
ID NO:29. In some embodiments, the host cell comprises one or more mutatioos in the endogenous YFH1 gene that results in a polypeptidc comprising a T I63P substitution, wherein the position of the substitution is relative to the amino acid positions of SEQ ID NO:31. In some embodiments. the host cell comprises one or more mutations in the endogenous NFS1 gene that results in a poly peptide comprising at least MK amino acid substitution selected from the group consisting of Li 15W and E451tD, wherein the position of the substitution is relative to the amino acid positions of SEQ ID NO:33. in sonic embodiments, the host cell comprises a mutation in the endogenous ISU I gene that results in a poly:peptide comprising the amino acid substitution WIN. wherein the position of the substittnion is Math e to the amino acid positions of SEQ
ID NO=29: and a mutation in the endogenous YFH1 gene that results in a polypeptide comprising the amino acid substitution T163P. wherein the position or the substilutioa is nclatiµc to the amino acid positions of SEQ.
ID NO:31. In some embodiments. the mutation is homozygous. In some embodiments. the mutation is heterozygous.
1001221 In some embodiments, the host cell comprises (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism, iron uptake, iron utilization. and/or an iron/sulfur (FOS) cluster biosynthesis: and (hi at least one heterologous gene encoding a polypeptide having xylosc isomerase activity. In sonic embodiments, at least one heicrologous polypeptide having xy lose isomerase activity is a xylose isomerase. One having skill in the an would understand that any number of known xylosc isomemse sequences could be expressed in the host cell of the present invention. In some embodiments the xy lose isotnerase is a naturally occurring xylosc isomerase. In some embodiments, the xylosc isomerase is a recombinant polypeptidc. In some embodiments. the sylose isomerase is a chimeric poly peptide. In some embodiments. the sy lose iSOIIICIIISC is encoded by a nucleotide sequence that has at least 80% sequence identity with a nucleotide sequence selected front SEQ ID NOs: 2. 4. 6, 8. 10. 12, 11.
16. 18. 20. 22. 24. 26. and 28. In some embodiments, the .sylose isomerase is encoded by a nucleotide sequence that has at least 83% sequence identity with a nucleotide sequence selected front SEQ ID NOs: 2. 4. 6. 8. 10, 12. 14. 16, IS. 20. 22. 24. 26. and 28. In %Me embodiments. the ss lose isomensse is encoded by a nucleotide sequence that bas at leasi 85%
sequence identity with a nucleotide sequence selected from SEQ ID NOs: 2. 4.
6. 8. 10. 12. 14. 16, 18. 20. 22, 24. 26. and 28. In some embodiments, the 'lose isomerase is encoded by a nucleotide sequence that has at least 87% sequence identity with a nucleotide sequence selected front SEQ ID NOs: 2.
4. 6. 8. 10, 12. 14. 16, IL 20, 22. 24. 26. and 28. In some embodiments, the sylose isomerase is encoded by a nucleotide sequence that has at least 90% sequence identity with a nucleotide sequence selected from SEQ ID
NOs: 2. 4,6. 8. 10. 12. 14. 16. IS.
20, 22, 24. 26. and 28. In sonic embodiments. the xy,lose isommase is encoded by a nucleotide sequence that has at leas1.91'% sequence identity with a nucleotide sequence selected from SEQ
ID NOs: 2. 4, 6. 8. 10. 12. 14. 16.
IX. 20, 22. 24, 26. and 28. In some embodiments, the xylose isomerase is encoded by a nucleotide sequence that has at least 92% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 2. 4,6. 8. 10, 12. 14.
16, IX, 20, 22. 24, 26, and 28. In some embodiments, the xi lost isomerase is encoded by a nucleotide sequence that has at least 93% sequence identity with a nuckotide sequence selected front SEQ ID NOs: 2. 4. 6. 8. 10. 12.
U. 16, 18, 20, 22, 24. 26. and 28. In URIC embodiments, the xylose isomerase is encoded by a nucleotide sequence that has at least 94% sequence identity si ith a nucleotide sequence selected from SEQ ID NOs: 2, I. 6, 8. 10. 12. 14, 16. 18, 20. 22. 24, 26, and 28. In some embodiments, the sy lose isomerase is encoded by a nucleotide sequence that bas at least 95% sequence identity with a nucleotide sequence selected from SEQ ID
NOs: 2,4,6. 8. 10. 12, 14, 16, IS, 20, 22. 24. 26. and 28. ln sonic embodiments. the xylose isomerase is encoded by a =Wide sequence that has at least 96% sequence identity with a nucleotide sequence selected from SEQ
ID NOs: 2. 4. 6. 8. 10. It 14, 16, 18, 20. 22, 24, 26. and 28. In some embodiments, the sylose isomerase is encoded by a nucleotide sequence that has at least 97% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 2. 4. 6, 8, 10, 12. 14. 16, 18. 20. 22. 24. 26, and 28. In some embodiments, the xylose isomerase is encoded by a nucleotide sequence that has at least 98% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 2. 4.6. 8, 10. 12, 14, 16. 18. 20. 22. 24.
26. and 28. In some embodiments, the sylose isomerase is encoded by a nucleotide sequence that has at least 99%
sequence identity with a nucleotide sequence selected from SEQ ID NOs: 2, 4, 6. 8. 10. 12, 14. 16, IX, 20. 22. 21, 26. and 2%. In some embodiments, the xylosc isomerase is encoded by a nucleotide sequence that has at least 100% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 2. 4, 6.8, 10. 12, 14, 16, 18. 20, 22, 24. 26, and 28, 1001231 In some embodiments, the xylose isomerase is encoded by a nucleotide sequence that has at least 80%
sequence identity with a nucleotide sequence selected from SEQ ID NOs: 2. 4.
6. 8. in. 12. 14, 16. 18. 20. 22.
24, 26. 28. 36, 38, 40. and 42 . In some embodiments. the vi lose isomerase is encoded by a nucleotide sequence that has at least 83% sequence identity with a nucleotide sequence selected front SEQ ID NOs: 2. 4.6. 8, 10. 12.
14. 16, IS. 20. 22. 24, 26, 28, 36, 38, 40, and 42 . In some embodiments. the sylose isomerase is encoded by a nucleotide sequence that has at least 85% sequence identity with a nucleotide sequence selected from SEQ ID
NOs: : 2, 4. 6. 8. 10, 12. 14. 16. 18, 20. 22, 24, 26. 28. 36, 38. 40. and 42.
In some embodiments. the xy lose isomerase is encoded b) a inicleotide sequence that has at least 87% sequence identity with a nucleotide sequence selected from SEQ ID NOs: : 2, 4.6. 8, 10. 12, 14, 16. 18. 20. 22.
14.26. 28, 36. 38. 40, and 42. In some embodiments, the xylose isomerase is encoded by a nucleotide sequence that has at !cast 90% sequence identity with a nucleotide sequence selected from SEQ ID NOs:: 2. 4. 6. 8. 10.
12. 14, 16. 18. 20, 22. 24. 26.28.
36. 38, 40. and 42. In some embodiments, the xylose isomerase is encoded by a nucleotide sequence that has at least 91% sequence identity with a nucleotide sequence selected from SEQ ID
NOs: : 2, 4. 6. 8, 10, 12. 14. 16.
I it. 20.22. 24. 26. 28. 36. 38. 40, and 42. In some embodiments the xylose isomerase is encoded by a nucleotide sequence that has at least 92% sequence identity with a nucleotide sequence selected from SEQ ID NOs: : 2. 4.
6. 8, 10. 12. 14. 16. 18. 20. 22. 24. 26. 28. 36. 38. 40, and 42. In some embodiments. the xylose isomerase is encoded by a nucleotide sequence that has at least 93% sequence identity with a nucleotide sequence selected from SEQ ID NOs: : 2. 4. 6. 8. 10. 12. 14, 16. 18, 20, 22. 24. 26. 28, 36, 38.
40. and 42. In some embodimerds.
the xylose isomerase is encoded by a nudeotide sequence that has at least 94%
sequence identity with a nucleotide sequence selected from SEQ ID NOs: : 2.4. 6, 8. 10,12. 14, 16, 111.
20. 22. 24, 26, 28, 36. 38. 40. and 42. In some embodiments, the xylem isomerase is encoded by a nucleotide sequence that has at least 95%
sequence identity with a nucleotide sequence selected from SEQ ID NOs: : 2. 4, 6, 8, 10. 12, 14. 16. 18. 20. 22.
24, 26. 28. 36. 38, 40. and 42. In some embodiments, the xylose isomerase is encoded by a nucleotide sequence that has at least 96% sequence identity with a nucleotide sequence selected from SEQ ID NOs: : 2. 4. 6. 8, 10.
12. 14, 16. 18. 20.22. 24. 26. 28, 36. 38, 40, and 42. In some embodiments.
the xylose isomerase is encoded by a nucleotide sequence that has at least 97% sequence identity isith a nucleotide sequence selected from SEQ ID
NOs: : 2. 4. 6, 8, 10. U. 14. 16, 18. 20. 22, 24, 26. 28. 16, 38, 40. and 42.
In some embodimems. the xylose isomerase is encoded by a nucleotide sequence that has at least 98% sequence identity with a nucleotide sequence selected from SEQ ID NOs: : 2. 4.6. 8. 10. 12. 14. 16. 18. 20, 22.
24,26, 28, 36. 18. 40, and 42 . In some embodiments. the xylose isomerase is encoded by a nucleotide sequence that has at least 99% sequence identits with a nucleotide sequence selected from SEQ ID NOs: : 2. 4. 6. 8,

10. 12. 14. 16. 18. 20, 22, 24. 26. 28.
36, 38. 40, and 42. In some embodiments. the .xy lose isomerase is encoded by a nucleotide sequence that has at least 100% sequence identity with a nucleotide sequence selected from SEQ ID
NOs: : 2. 4, 6. 8, 10, 12. 14. 16.
18, 20, 22, 24, 26, 28. 16. 38. 40, and 42.
1001241 In some embodiments, the xylose isomerase has an amino acid sequence that has at least 80% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1, 3. 5, 7. 9,

11. 13. IS. 17. 19.21. 23, 25. and 27. In sonic embodiments, the xylose isonemse has an amino acid sequence that has at least 83% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1 , 3 . 5 , 7.9. II. 13. IS. 17. 19. 21. 23. 25. and 27. In some embodiments, the xy lose isomerase has an amino acid sequence that has at least 85% sequence identity %%int an amino acid sequence selected from SEQ ID NOs: 1,3. 5, 7, 9, 11, 13. 15. 17. 19,21. 23.25. and 27. In some embodiments, the n lose isomerase has an amino acid sequence that has at least 87% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1. 3, 5. 7,9, 11, 13. 15, 17, 19, 21, 23, 25, and 27. In some embodiments, the xylose isomenisc has an amino acid sequence that has at least 90% sequence identity ith an amino acid sequence selected from SEQ ID NOs: 1 3 7. 9.
II. 13. 15, 1.7. 19. 21. 23. 25. and 27. In sonic embodiments, the x.) lose isomerase has an amino acid sequence that has at least 91% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1, 3. 5, 7,9, 11, 13. 15. 17. 19.21. 23,25. and 27. In some embodiments. the xylose isomerase has an amino acid sequence that has at Itt1S1 92% sequence ' identity with an amino acid sequence selected from SEQ ID NOs: 1. 3.5. 7.9.
II. 13. 15. 17. 19. 21, 23. 25. and 27. In sonic embodiments. the xlose isomerase has an amino acid sequence that has at least 93% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1. 3.5. 7.9. I
I. 13. 15. 17, 19, 21, 23. 25. and 27. In some embodiments. the xy lose isomerase has an amino acid sequence that has at least 94% sequence ideniit uith an amino acid sequence selected from SEQ ID NOs: I. 3. 5. 7. 9. I
1. 13. 15. 17, 19. 21.23. 23. and 27. In SCIMC embodiments. the xylosc isonerase has an amino acid sequence that has at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: I. 3, 5. 7.9.
11. 13. 15. 17. 19. 21. 21. 25. and 27. In some embodiments, the xylose isomerase has an amino acid sequence that has at least 96% sequence identity with an amino acid sequence selected from SEQ ID NOs: I. 3. 5. 7. 9, 11. 13. 13. 17. 19. 21. 23, 25. and 27. hi sonic embodiments, the xylosc isomerase has an amino acid sequence that has at least 97% sequence identity uith an amino acid sequence selected from SEQ ID NOs: I. 3. 5, 7, 9, 11. 13. IS. 17. 19. 21. 23. 25, and 27. In wine embodiments, the xy lose isomerase has an amino acid sequence that has at least 913% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1.3. 5. 7. 9. I
I. 13, 15. 17, 19. 21. 23.25. and 27. In seine embodiments, the xylose isomerase has an amino acid sequence that has at least 99% sequence identity with an amino acid sequence selected from SEQ ID NOs: I. 3. 5. 7. 9.
11, 13, IS, 17. 19. 21. 23, 25, and 27. In some embodiments, the xylose isornerase has an amino acid sequence that has at least 10% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1. 3, 5. 7, 9, 11, 13, IS, 17, 19, 21. 23. 25, and 27.
1001251 In sonic embodiments, the xylosc isonerase has an amino acid sequence that has at least 80% sequence identity with an annno acid sequence selected from SEQ ID NOs. I. 3 . 3 . 7.9.
11, 13. IS. 17, 19, 21. 21. 25. 27, 35, 37. 39. and 41. In sonic embodiments. de 4Iosc isomerase has an amino acid sequence that has at least 113%
sequence identity with an amino acid sequence selected from SEQ ID NOs: 1. 3.
5, 7.9. 11. 13, 15, 17, 19, 21, 23, 2.5, 27. 35, 37. 39. and 41 . In some embodiments. the x'. lose isomerase has an amino acid sequence that has at least 115% sequence ideivits with an amino acid sequence selected from SEQ
ID NOs: I. 3. 5. 7.9. 11. 13. IS.
17, 19. 21, 23. 25, 27. 35, 37, 39. and 41. In sonic embodiments, the ylose isomerase has an amino acid sequence that has at least 87% sequence identity with an amino acid sequence selected from SEQ ID NOs: I. 3, 5, 7. 9, 11, 13, 15, 17, 19. 21. 23. 25. 27. 35. 37, 39. and 41. In some embodiments, the xylose isomerase has an amino acid sequence that has at least 90% sequence identity with an amino acid sequence selected from SEQ ID
NOs: I. 3. 5. 7. 9, 11, 13. 15. 17, 19. 21,23, 25, 27, 35. 37. 39. and 41. In some embodiments_ the xylose isomerase has an amino acid sequence that has at least 91% sequence identit isith an amino acid sequence selected from SEQ ID NOs: 1. 3. 5. '7. 9. 11. 13. 15, 17. 19. 21. 23, 25. 27.
35, 37. 39. and 41 43. In some embodiments, the sylose isomerase has an amino acid sequence that has at least 92% sequence identity with an amino acid sequence selected from SEQ ID NOS: I. 3.5. 7,9, It. 13. 15. 17. 19.
21. 23. 25_ 27. 35. 37. 39, and 41. In some embodiments. the sy lose isomerase has an amino acid sequence that has at least 93% sequence identity with an amino acid sequence selected from SEQ ID NOs: I. 3. 5, 7, 9, 11. 13. IS. 17. 19.21. 23. 25.27.
35, 37,39. and 41. In some embodiments. Or xylose isomerase has an amino acid sequence that has at least 94%
sequence identity with an amino acid sequence selected from SEQ ID NOs: IL 3.
5. 7, 9. 11. 13. IS. 17. 19. 21_ 23. 25,27, 35, 37, 39. and 41. In some embodiments, the xy lose isotnerase has an amino acid sequence that has at least 95% sequence identity with an amino acid sequence selected from SEQ
ID NOs: 1. 3, 5. 7.9, 1 1. 13, 15, 17. 19, 21, 23, 25, 27, 35, 37, 39, and 41. In sonic embodiments. the xy lose isomerase has an amino acid sequence that has at least 96% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1, 3.

WO 2(116/024215 PCT/I B2015/056101 5. 7.9. II. 13, 15. 17. 19.21. 23. 25. 27, 33.37, 39, and 41. In some embodiments. the x lose isomcrase has an amino acid sequence that has at least 97% sequence identit) with an amino acid sequence selected from SEQ 113 NOs: I. 3, 1. 7. 9. 11, 13. 13, 17, 19, 21. 23. 25, 27. 35. 37, 39. and 41. In some embodiments, the xylose isomerase has an amino acid sequence that has at le.ast 98% sequence identity with an amino acid sequence selected from SEQ ID NOs: I. 3. 5. 7, 9. 11. 13. IS, 17. 19. 21, 23. 25. 27.
35. 37. 39. and 41 43. In some embodiments. the xylose isonterase has an amino acid sequence that has at least 99% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1. 3. 5. 7, 9. II. 13. IS. 17, 19.21. 23. 25. 27, 35, 37. 39. and 41. In some embodiments, the xylose isomerase has an amino acid sequence that has at least 10% sequence identity with an amino acid sequence selected from SEQ ID NOs: I. 3. 5, 7, 9.
11. 13. 15, 17, 19. 21. 23. 25. 27, 35, 37. 39. and 41.
1001261 In some embodiments, the host cell comprises (a) one or mutation in the endogenous YFH I gene that results in a polypeptide comprising a TI.63P substitution: and (b) at least one heterotogous gene encoding a polypeptide having xylose isomerase activity, wherein the polypeptide has an amino acid sequence at least about 80%. at least about 83%. at least about 85% at least about 87%. at least about 90%. at least about 91%. at least about 92%. at least about 93%. at least about 94%. at least about 95%. at least about 96%, at least about 97%. at about least 98%, at about least 99% or about 100% identical to the amino acid sequence of SEQ ID NO.1. In some embodiments, the host cell comprises (a) a deletion or disruption of GRE3 and/or YPR1: (b) one or more mutations that correlate midi an increase in the expression or up-regulation of one or more of XKSI. RK11.
R PE I TALI, Pla141 and/or PCIM2; (e) one or mutation in the endogenous YFH1 gene that results in a poly peptide comprising a T163P substitution: and Id) at least one heterologous gene encoding a polypeptide having x3, lose 'sometime activity, wherein the polypeptidc has an amino acid sequence at least about 80% at least about 83%, at least about 85%. at least about 87%, at least about 90%.
at least about 91%. at least about 92%. at least about 93% at least about 94%. at least about 95%. at least about 96%. at least about 97%, at about least 98%. at about least 99%. or about 100% identical to the amino acid sequence of SEQ ID NO:l. In some embodiments. the host cell can be cultured in a medium supplemented with iron.
In some embodiments, the host cell can be cultured under conditions that facilitate and/or stimulate the uptake of iron by the host cell. In some embodiments. the host cell can be cultured under conditions that hinder, prevent, block, and/or decrease the export of iron from the host cell.
1001271 In some embodiments, the host cell comprises more than one copy of the poly nucleotide encoding the polypeptide having xylosc isomerase activity. In sonic embodiments, the host cell comprises two copies. three copies. four copies. five copies. six copies. seven copies, eight copies. nine copies. ten copies. eleven copies, at least twelve copies. at least fifteen copies, or at least twenty copies of the polynucleotide encoding the poly-peptide having xyloscisonierase activity.
(00128j In some embodiments, the polynucleotide can be present in a vector. In some embodiments, the host cell can comprise the p0.nueleotide within a vector. In some embodiments, the vector is a plasmid. In some embodiments, the host cell can express the polynucleotide from the vector. In some embodiments, the pols nucleotide can he incorporated into the gennine of the host cell. lit some embodiments, the host cell is a fungal cell. lit some embodiments, the host cell is a yeast cell. In some embodiments. the host cell is a S.
cerevisiac cell.
1001291 Certain embodiments of the present invention describe methods for producing a fermentation product.

In certain embodiments, the recombinant host cell comprising the polynucleotide or the polypeptide and a mutation in one or more genes encoding a protein associated with iron metabolism is contacted with a carbon source. In some embodiments, the host cell comprises a mutation in one or more genes encoding a protein associated with iron metabolism, and the host cell is contacted with a carbon source and an exogenous source of a polypeptide having xylose isomerase activity. In certain embodiments, the carbon source comprises xylose. In certain embodiments, xylose is the sole source of carbon in the carbon source.
In certain embodiments, a fermentation product is produced by contacting the host cell with the carbon source. In certain embodiments, the fermentation product is recovered. In certain embodiments, the fermentation product is selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, hydrogen, butyric acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, acetone, isopropyl alcohol, butanol, a 13-lactam, an antibiotic, cephalosporin, or a combination thereof. In certain embodiments, the fermentation product is ethanol.
IV. Codon-Optimization [001301 In some embodiments, the nucleotide sequence of the one or more polynucleotides disclosed in the present invention are codon-optimized for expression in a fungal host cell. In some embodiments, the nucleotide sequence of the polynucleotide is codon-optimized for expression in a yeast host cell. In some embodiments the nucleotide sequence of the polynucleotide is codon-optimized for expression in S. cerevisiae. Codon-optimized polynucleotides can have a codon adaptation index (CAI) of about 0.8 to 1.0, about 0.9 to 1.0, or about 0.95 to 1Ø
100131] In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the "codon adaptation index" or "CAI,"
which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism. The Codon Adaptation Index is described in more detail in Sharp and Li, Nucleic Acids Research 15:1281-1295 (1987).
1001321 The CAI of codon-optimized sequences used in the present invention corresponds to from about 0.6 to about 1.0, from about 0.7 to about 1.0, from about 0.8 to about 1.0, from about 0.9 to about 1.0, from about 9.5 to about 1.0, or about 1Ø A codon-optimized sequence can be further modified for expression in a particular organism, depending on that organism's biological constraints. For example, large runs of "As" or "Ts" (e.g., runs greater than 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can be removed from the sequences if these are known to effect transcription negatively. Furthermore, specific restriction enzyme sites can be removed for molecular cloning purposes. Examples of such restriction enzyme sites include Pad, Ascl, BamHI, Bg111, EcoRJ and Xhol.
Additionally, the DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of ten bases or longer, which can be modified manually by replacing codons with "second best" codons, i.e., codons that occur at the second highest frequency within the particular organism for which the sequence is being optimized 1001331 Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene.
Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code" which shows which codons encode which amino acids is well known to one of skill in the an. As a result, many amino acids arc designated by more than one codon. For exampk. the amino acids alaninc and prolate arc coded for by four triplets. scrine and argininc by six. whereas tryptoplin and methionine are coded by just one triplet. This degenetaey allows for DNA base composition to % ary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.
1001341 Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias. differences in codon usage between organisms.
is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA). which is in turn believed to be dependent on. inter alia. the properties of' the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected ilINAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly. genes can be tailored for optimal gene expression in a given organism based on codon optimization.
1001351 Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage.
Codon usage tables and codon-optimizing programs are readily. available, for example. at littp://www.kazusa.or.jpicodon/ (visited July IS.
2014), and these tables can be adapted in a number of ways. See. e.g..
Nakamura. Y.. et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000." Nucl. Acids Res. 28:292 (200o).
1001361 By utilizing one or more available tables. one of ordinal) skill in the an can apply the frequencies to any given poly-peptide sequence. and produce a nucleic acid fragment of a codon-optimized coding region which encodes the poly peptide. hut which uses codons optimal for a given species.
Codon-optimizix1 coding regions can be designed by various different methods known to one having ordinary skill in the an.
1001371 In certain embodiments. an entire polypcptide sequence. or fragment.
variant. or derivative thereof is codon-optimized by any. method known in the art. Various desired fragments.
variants or derivatives arc designed. and each is then codon-optimized individually. In addition.
partially codon-optimized coding regions of the present invention can be designed and constructed. For example. the inentioui includes a nucleic acid fragment of a codon-optimized coding region encoding a poly-peptide in which at least about 1%. 2%. 1% 4%, 5%. 10%. 15%, 20%, 23%, 30111. 35%. 40%. 45%, 50%,' 53%, 60%, 65%, 70%. 75%.
80%, 8.5%. 90%. 95%. or 100% of the codon positions have been codon-optimized for a given species.
That is, they contain a codon that is preferentially used in the genes of a desired species. e.g.. a yeasi species such as S. ceirvisiae. in place of a codon that is normally used in the native nucleic acid sequence.
1001381 In additional embodiments, a full-length poly peptide sequence is codon-optimized for a given species resulting in a codon-optimized coding region encoding the entire poly.peptide.
and then nucleic acid fragments of the codon-optimized coding region, which encode fragments. variants, and derivatives of the poly.peptide are made from the original cockm-optimized coding region. As would be well understood by those of ordinary skill in the art, if codons have been randomly assigned to the full-length coding region based on their frequency of use in a given species, nucleic acid fragments encoding fragments. variants, and derivatives would not necessarily. be fully codon-optimized for the given species. However, such sequences are still much closer to the codon usage of the desired species than the native codon usage. The advaniage of this approach is that sy nthesizing codon-optunitcd nucleic acid fragments encoding each fragment. ariant. and derivative of a given poly peptide.
although routine. %%mild be time consuming and Nvould result in significant expense.
1001391 In some embodiments, one or MOW of the donor parent polynneleotide sequences are codon-optimited for expression in yeast. In some embodiments, the chimeric poly nucleotide is codon-optimized for expression in yeast.
V. Methods of-Producing Ethanol 1001401 Certain aspects of the present invention are directed to methods of producing a fermentation product. In some embodiments of the invention, the recombinant host cell is used to produce a fermentation product front a cellulosic or lignoccIltdosie material. In sonic embodiments. the fermentation product is ethanol, lactic acid. 3-hydroxy-propionic acid. hydrogen, butyric acid. acrylic acid, acetic acid.
succinic acid, citric acid. malic acid.
fumaric acid, an amino acid. 1.3-propanc-diol, ethylene. glycerol. acetone, isopropyl alcohol. butanol. a p-laciam, an antibiotic, a cephalosporin. or a combination thereof. In some embodiments, the cellulosic or lignocellulosic material is insoluble cellulose, crystalline cellulose.
pretreated hardwood, paper sludge. pretreated corn stover, pretreated sugar cane bagasse. pretreated corn cobs. pretreated switchgrass. pretreated municipal solid waste. pretreated distiller's dried gmins pretreated wheat straw. corn fiber. agave. or a combination thereof.
1001411 One aspect of the invention is directed to a composition comprising a lignoccllulosic material and a recombinant yeast host cell comprising at least one poly peptide having xylose isomerase activity and comprising a mutation in a gene encoding a protein associated with iron metabolism.
Another aspect of the invention is directed to it media supernatant generated b) incubating a recombinant =yeast host comprising as Icas-1 one poly peptide having X) lose isomerase activity and contpnsing a mutation in a gene encoding a protein associated with iron metabolism with a medium containing lose as the only carbon source.
In some embodiments, the medium comprises a celltilosic or lignocellulosic material. In some embodiments, the cellulosic or lignocellulosic material is insoluble cellulose. ors stannic cellulose.
pretreated hardwood. paper sludge. saw mill or paper mill discards. pretreated corn stoner. pretreated sugar cane bagasse.
pretreated corn cobs. pretreated switehgrass. pretreated municipal solid N+astc. pretreated dried grains.
pretreated wheat straw, corn fiber. agave. or a combination thereof.
10411142] In some embodiments, a fermentation product is produced by a method comprising contacting a recombinant host cell of the present invention with a carbon source, wherein the carbon source comprises xylose.
In sonic embodiments. thc fermentation product is selected front the group consisting of ethanol, lactic acid.1-hydroxy-propionic acid. hydrogen. butyric acid, acrylic acid, acetic acid, succinic acid, citric acid. malic acid, fuanaric acid, an amino acid. 1.3-propanc-diol. ethylene. glycerol. acetone.
isopropy I alcohol. Milano!. a fi-laciam. aim antibiotic, and a cephalosporin. In some embodiments. the fermentation product is ethanol. In some embodiments, the fennentation product is recovered.
1001431 Certain aspects of the present invention arc directed to a method of producing ethanol comprising contacting a source material comprising xylose with a host cell of the present invention. In some embodiments the host cell heterologously expresses a poly-peptide having xvlose isomerase activity. In some embodiments the host cell further comprises a mutation in one or more genes encoding a palypcptide that is associated with iron metabolism.
100144] In some embodiments, the source material is a cellulosic bionmss. In some embodiments, the source material is a lignoccIltdosic biomass. In some embodiments, the source material is selected from the group consisting of insoluble cellulose. crstalIine cellulose. pretreated hardwood, softwood, paper sludge, newspaper.
sweet sorghum. pretreated corn siov cr. pnareated sugar cane bagasse.
pretreated corn cobs. pretreated switchgrass. pretreated municipal solid waste. pretreated distiller's dried grains. pretreated wheat snow. ricc straw. nut shells, banana waste, sponge gourd fibers, corn fiber. agave, trees. corn stover. wheat straw. sugar cane bagasse. so itchgrass. and combinations thereof In some embodiments, the source material is corn stover EXAMPLES
1001451 The invention now being generally described. it will be more readily understood by reference to the following examples. which are included merely for purposes of illustration of certain aspect and embodiments of the present invention, and are not intended to limit the invention.
Example 1 - S. cerevisiae background strain 1001461 A strain of S. cerevisMe was created that was suitable for the testing or functional xylosc isomerases.
The GRE3 locus of an industrial yeast strain was replaced with expression cassettes for the pentose phosphate pathway genes RPEI. RKII. TKL I. and TALI as well as the native S. cerevisiac ulokitmse XKSI (Figure 1).
Example 2 - Identification of non metabolism related genes mutated in N1 lose utilizing strains 1001471 Specific mutations in three native S. cerevisiae genes (ISU I. YFH I .
and NESI) were identified that significantly improve performance of XI xylose engineered strains. The mutations were identified by reverse engineering several strains adapted for improved growth rate on xylosc media.
The adapted strains %%ere denved from strains engineered to express an exogenous X1 and to ovcrexpress the native genes XKS. RKII. RPE I
TALI. and TKL I Two strains were adapted that differed in the native GRE3-1-locus, with one strain having n deletion of the endogenous GRE3. The mutations can be directly engineered into a strain providing the performance improvements usually obtained via adaptation. The directed engineering of these mutations saves the time and uncertainty associated with strain adaptations. These mutations can benefit strains engineered with various Xls (sec Figures Sand 6).
Example 3 - Mutations in YFH11, 'SUL and NFS I improve growth on xy lose 1001481 Strains were grown on YPX media (yeast extract. peptone, and x) lose) under anaerobic conditions in a Biock plate reader. 0D600 measurements were used to detemiine changes in cell density over time (-48hrsi (Figure 3). Xylose Utilizing Strains (XUS) I and 2 are strains engineered to utili/e xy lose but %N ithout mutations in YFH I. ISO!. or NFS1. XUSI-1 and XUSI -2 strains were adapted for improved growth on xy lose originating from strain XUSI. Strain XUS2-I was adapted for improved growth on xylose originating from strain XUS2.
Genone sequencing revealed mutations in iron-sulfur cluster related genes in the adapted strains XUS I -1 (YFH1), XUSI -2 (NFS1) and XUS2-1 (ISU I ). Direct genetic engineering to reven the mutations to the wild ts pc alleles (XUSI-I ->Y12HI wi. XUS2-1 XUS1-2 ->NPS1 wt) decreased x) lose growth. matching the original parent strains. Direct genetic engineering of the iron-sulfur mutations into the parent strains (XUS I ->YFH I T163P, XUS2 ->1SUI D71N. XUS I ->NESI L115W) resulted in improved xylosc growth matching the adapted strains with the same parent and mutation. The 1SU1 D7IN imitation was direct engineered as a hetemz) gotc to match the mutation found in the adapted strain XUS2-1.
Example 4 - Homozygousing the ISU1.137 IN mutation improves growth on xylose 1001491 Strains were grown on YPX media (yeast extract, peptone, and xylose) under anaerobic conditions in a Blotch plate reader. OD600 measurements were used ID determine changes in cell density over time (--41ilu5) (Figure 4A). The negative control is a strain that is unable to grow on xylose. Adapted strain XUS2-1 is .31 heterozygous at the ISU1 locus. XUS2-I genetically engineeted with two mutant alleles of ISUlD71N (XUS2-1 +1St.' (* bomb) exhibits improN cd growth on xy lose relative to the original heterozygote XUS2-1. Engineering the original parent strain with two mutant alleles of ISU1D7IN (XUS2 +ISUI*
hotno) results in improved xy lose growth equivalent to the XUS2-1 ISUID7IN homozygote.
Example 5 - The homozygous ISUID7IN mutation improves growth of the XUSI GRE3+
parent et rain 1001501 Strains were grown on YPX media (cast extract. peptone. and x) lose) under anaerobic conditions in a Biotek plate reader. 0D600 measurements were used to determine changes in cell density over time (--48hrs) (Figure 4B). The negative control is a strain that is unable to grow on xylose. The ISLIID7IN mutation was identified as a heterozygous mutation in an adapted xylose-utilizing strain with GRE3 deleted (XUS2-1). Direct engineering of the ISUID7IN heterozygous mutation into the GRE3+ xylose strain XUSI did not improve x-ylose growth (data not shown). Engineering XUS1 strain with two mutant alleles of ISUlD7 IN (XUSI +ISUI*
home) results in significantly improved xylose growth equivalent to the XUS2 directly engineered ISUID7IN
homozygote (XUS2 +ISU I* horno). Straw XUS I-1 is an adapted version of XUSI
containing a homozygous mutation in YFH1. XUSI -I directly engineered homozygous ISUID7IN exhibits decreased performance.
Example 6 - The YFH IT I63P mutation improves growth of the yeast strains heterologously expressing N ariousXls 1001511 Strains were grown on YNBX minimal media, and the 0D600 was measured following 4K hours of aerobic growth at .35 C (Figure 5). Various Xis were expressed on plasmids within the industrial host strain used for the chimeric XI library (black bars) or the host strain plus the YFH I
1163P Fe/Su cluster mutation (hashed bars). Eight colonies from each transformation were inoculated into YNBX
media. Nearly all of the Xls that generated growth above the negative control, which lacked an XI, showed a benefit from the presence of the YFH I mutant allele.
1001521 In a second set of experiments, strains were grown on YPX media (yeast extraci peptone, xylose) under anaerobic conditions in a Biotck plate reader at 35 C. 0D600 measurements were used to determine changes in cell density over time (-411 hours) (Figure 6 A and B). The negative control is a strain unable to grow on xy lose.
Figure 6A shows strains containing the wild type allele of YFHI. Figure 6B
shows strains containing the YFH11163P allele. All of the Xls tested using this genontic integration format showed significantly improved growth on xylosc with time YFHIT163P allele present. CX355 = chimeric xylose isomerase 355 = CX1224 =
chimeric xylose isomerase 1224. Ad = Abiotrophia defectiva. 131 = Bactcriodes thetaioatomicron. Pc =
Piromyces. Is = Lachnoanaerobaculum saburreum Example .7- Mutations in AFT' and CCC1 improve xylosc growth 1001531 Strains were grown on YPX media (yeast extract. peptone, and xylosc) under anaerobic conditions in a Biotck plate reader. 0D600 measurements were used to determine changes in cell density over time (--413111's) (Figure 7). The negative control is a strain that is unable to grow on xylosc.
Xy lose utilizing strain (XUS) is a strain engineered to utilize xy lose XUS I-1 strain was adapted for improved growth on 'Mose originating from strain XUS I and was found by gnome sequencing to contain a mutation in iron-sulfur cluster related gene YFHI: XUS1-1 serves as a positive control. Direct engineenng of the AF1'I-1UP
allele into the XUSI wain (XUS1 +AFTI-1UP) slightly improved growth on xylosc. Direct engineering of the AFT1-1UP allele into and deletion of both endogenous copies of CCC1 in the XUSI strain (XUSI +AFT1-11113. eceld) result in significantly improved xylose growth close to that of the XUSI-1 strain.
Example 8- Addition of iron improves growth on xylose [00154] Strains were grown on SP1 media (yeast nitrogen base with amino acids, tri-sodium citrate, glucose, xylose) under anaerobic conditions in serum bottles. Samples were taken and measured for ethanol, xylose and glucose concentrations over time (-65 hours) (Figure 8).
Xylose Utilizing Strain 2 (XUS2) is engineered to utilize xylose. Strain XUS2-1 was adapted for improved growth on xylose originating from X1JS2. Genome sequencing revealed mutations in iron-sulfur cluster related gene ISU1 in strain XUS2-1. Samples indicated as "H-iron" were supplemented with iron at the start of the fermentation.
The strains consumed all of the glucose at similar rates during the first ¨18 hours of the fermentation and produced similar amounts of ethanol with no difference seen with the addition of iron. In contrast, the addition of iron significantly improved the rate of xylose utilization as seen in the increased ethanol production between 18 and 65 hours. The increased xylose utilization (and subsequent ethanol production) was seen for both strains with and without the mutations in the iron-sulfur cluster related genes.
Example 9 ¨ Iron addition enables significant activity of xylose isomerase in vitro [00155] Xylose isomerase functions as a tetramer with the binding of two divalent cations per subunit essential for enzyme activity. Mg2+, Mn2+, Co2+, and Fe2¨ ions activate the enzyme (Waltman et al.
Protein Engineering, Design & Selection, 2014, p. 1-6). Using an in vitro enzymatic assay, the addition of Fe2+ was found to result in significantly more xylose isomerase activity than the addition of Mg2+ (Figure 9). The protocol was essentially the same as described in Zou et al (Metabolic Engineering. 14, 2012, p.
611-622) with the exception of the use of three different buffers for the assay which varied in the absence or presence of the divalent metals Mg2 I or Fe2+. A cell extract was made from strain XUS1 which expresses the Bacteriodes thetaiotaomicron xylose isomerase. The cell extract was combined with Tris buffer H /- divalent metals, NADH, and sorbitol dehydrogenase. The assay was initiated with the addition of xylose and the reaction was monitored for 2 minutes at 340 nm to determine the initial rate. The reactions were performed under inert atmosphere and reducing conditions to deter oxidation of Fe2+ to Fe3+. One unit of activity is equal to lurnol NADH oxidized/mm/ml, which corresponds directly with the consumption of the xylose that is added to initiate the reaction.
[00156] Cancelled [00157] Following are particular embodiments of the disclosed invention:
100158] El. A recombinant yeast cell comprising (a) at least one heterologous gene encoding a protein associated with iron metabolism and/or one or more mutations in one or more endogenous gene encoding a protein associated with iron metabolism; and (b) at least one heterologous gene encoding a polypeptide having xylose isomerase activity.
[00159] E2. The recombinant yeast cell of El, wherein the at least one heterologous gene encoding a protein associated with iron metabolism and/or the one or more mutations in one or more endogenous gene encoding a protein associated with iron metabolism confers on the recombinant yeast cell an increased ability to utilize xylose as compared to a similar yeast cell lacking the one or more mutations.
[001601 E3. The recombinant yeast cell of El or E2, wherein the one or more mutations is a heterozygous mutation.
1001611 E4. TIE recombinant yeast cell of El or E2. wherein the one or tare mutations is a hoinoty gous mutation.
1001621 E. The recombinant yeast cell of any one of El -E4. wherein the recombinant yeast cell is a member of a genus selected from the group consisting of Saccharotnyces. Klinveromyces.
Candida. Pichia.
Schib5saccharonlyces. Hansenula. Klocckera. Schwannioniyees. and YaTTOIVia 1001631 E6. The recombinant yeast cell of claim E3. wherein the recombinant yeast cell is a member of a species selected front the group consisting of Saccharontyces cerevisiae.
Saccharoinyces buldcri. Saccharomyces exiguus. Saccharomyces uvarum Saccluiromyces diastaticus. Candida krusei.
Klocckera hictis. Klocckera marsianus. and Klocckera fragilis.
(001641 E. The recombinant yeast cell of claim ES. wherein the recombinant yeast cell is a member of a species selected from the group consisting of Saccharomyces cerevisiac.
Saccharomyces bulderi, Saccharomyces cxiguus, Saccharomyces uvanim. Saccharonnves diastaticus. Kloeckera lactis.
Klocckera marxianus. and Klocckera fragil is.
1001651 Eli. The recombinant yeast cell of an one of El -E7. wherein the recombinant yeast cell is S. cereviscie.
1001661 En. The recombinant yeast cell of any one of El-E4, wherein the one or more mutations in an endogenous gene is in a gene selected from the group consisting of ISU1. YFH
I. NFS1. AF1'1. AFT2. YAPS.
FRA I. FRA2. GREX3, GREX4. CCCI. and any combination thereof.
1001671 EH). The towsubinant yeast cell of E9, herein the one or more mutations is a sttbslitution of at !cast one nucleotide.
11)(11681 Eli. The recombinant y east cell of E10. wherein the recombinant yeast cell comprises one or more mutations in the endogenous ISU I gene that results in a polypeptide comprising at least one amino acid substitution selected front the group consisting of 1)71N. D7lG, and S98F. w herein the position of the substitution is relative to the amino acid positions of SEQ 11) NO:29.
1001691 E12. TIE recombinant yeast cell of Eli) or Eli. wherein the recombinant yeast cell comprises one Of more mutations in the endogenous YFH1 gene that results in a polypeptide comprising a T163P substitution.
w herein the position of the substitution is relative to the amino acid positions of SEQ ID NO:31.
1001701 El 1. The recombinant yeast cell of any one of E10-E1 2. wherein the recombinant yeast cell comprises one or more mutations in the cndogcnous NFS1 gene that results in a polypeptidc comprising at least one amino acid substitution selected front the group consisting of LI15W and E458D.
wherein the position of the substitution is relative to the amino acid positions of SEQ ID NO:33.
1991711E14. The recombinant yeast cell of aims one of E9-E13. wherein the recombinant yeast cell comprises a mutation in the endogenous AFTI gene that results in increased Aftl activity.
1001721 15. The recombinant yeast cell of any one of 9-Elk wherein the recombinant yeast cell comprises a mutation in the endogenous AFT2 gene that results in increased A112 activity.
100.1731 16. The recombinant yeast cell of any one of E9-E13, wherein the recombinant yeast cell comprises one or more mutations in one or more endogenous genes selected from FRA1.
FRA2. GREX3. and GREX4;
wherein the one or more mutations results in increased activity of Aftl and/or A02: and/or wherein the one or more mutations results in increased expression of one or more genes regulated by Aftl and/or A112.
1001111 E17. The recombinant east cell of 16. wherein the recombinant yeast cell further comprises a WO 20161024215 PC17182015/0.56101 mutation in an endogenous gene selected from the group consisting of YAP5 and CCC I.
[001751 E18. The recombinant yeast cell of E17, wherein the recombinant yeast cell comprises a deletion or disruption of YAP5 or CCC I .
1001761 E19. The recombinant ,yelISI cell of any one of El-E18, wherein the heterologous gene (a) is selected from the group consisting of AFT], AFT2, and onhologues and combinations thereof.
1001771 E20. The recombinant yeast cell of any one of EI-E18. wherein heterologous gene (a) encodes a protein that increases the activity of Aftl and/or Aft2 and/or increases the expression of AFr 1 and/or AFT?.
1001781 E21. The recombinant yeast cell of E1.8. wherein the hetcrologous gene (a) encodes a protein that suppresses or inhibits the activity and/or expression of a protein that suppresses or inhibits the activity of Aft!
and/or Aft2 and/or suppresses or inhibits the expression of AFT! and/or .AFT2, 1001791 E22. The recombinant yeast cell of any one of E 1 -EIS, wherein the bcterologous gene (n) encodes a target of Afil and/or Aft2, 1001801 E23, The recombinant yeast cell of any one of El -E18. wherein the heterologous gene (a) encodes a polypeptide having iron transport activity.
1001811 E24. The recombinant yeast cell of any one of E]-E23. wherein the haerologous gene (a) is constitutively espressed.
1001821 E25. The recombinant yeast cell of any one of El-E24. wherein the heterologous gene (b) encodes a sylose isomerase enzyme.
1001831 E26. The recombinant yeast cell of E25, wherein the heterologons gene (h) encodes a polypcptide having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ
ID NOs: L3, 5,7. 9. Ii, 13. 15, 17. 19.. 21, 23. 25, 27, 35, 37. 19, and 4,1.
1901.841 E27. The recombinant yeast cell of E25, wherein the heterologous gene (b) encodes a polypeptide having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ
ID NOs: 1, 3, 5, 7, 9, 11, 13, 15. 17, 19, 21,23, 25, awl 27, 1001851 E28. The recombinant yeast cell of E26. wherein the hcterologous gene (b) encodes a polypeptide having it least 85% sequence identity with an amine acid sequence selected from the group consisting of SEQ
1..D NOs 1. 3, 5. 7, 9. 11.13, 15, 17. 19, 2.1,23. 25. 27, 35, 37, 39, and 41.
100186.1 E29, The recombinant yeast cell of E27, wherein the hetemlogous gene (b) encodes a polypeptidc having al least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ
ID NOs: 1, 3, 5, 7, 9. Ii, 13, 15, 17, 19, 21.23. 25. and 2.7, 1001871 E30. The recombinant yeast cell of E28, wherein the hcternlogous gene (b) encodes a polypeptide having at least 90`;--ii sequence identity with an amino acid sequence selected from the group consisting of SEQ
1.1) NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27. 35, 37, 39, and 41, 1001881 E31, The recombinant ;yeast cell of .E29. wherein the hcterologous gene (b) encodes a polypeptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ
ID NOs: 1. 3, 5, 7. 9. 11. 13. 15, 17. 19..21.23. 25, and 27, 1001891 E32. The recombinant yeast cell of E30, wherein the heterologotts gene (b) encodes a polypeptidc haying al least 95% sequence identity with an amino acid sequence selected front the group consisting of SEQ
ID NOs: 1, 3, 5, 7, 9, ii. 13, 15..17, 19. 21,23. 25, 27,.35, 37, 39, and 41.
1001901 E33. The recombinam yeast cell of EM. wherein the hetcrologous gene (b) encodes a polypeptide WO 2(116/024215 P(171132015/056101 having at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ
ID NOs: I, 3, 5, 7, 9, II, 13, 15, 17, 19.21. 23, 25, and 27.
1001911 E34. The recombinant yeast cell of En. wherein the beterologous gene (b) encodes a polypemide having 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:
1,1, 5, '7. 9, 11. 13. IS. 17.19, 21, 23, 25, 27 Ii 37, 39, and 41.
1001921 E35. The recombinant yeast cell of E33. wherein the licterologour gene (b) encoder a polypeptide having 100% sequence identity with an amino acid sequence se lect.ed from the group consisting of SEQ ID NOs:
1 3 5 7. 9, 1.1..13. 15. 17.19, 21, 23,25, and 27.
1001931 E36. The recombinant yeast cell of any one of El-E35, wherein the recombinant yeast cell further comprises at least one genetic modification of one or more endogenous genes encoding a protein of the pentose phosphate pathway.
1001941 E3-7, The recombinant yeast cell of E36, wherein the recombinant yeast cell comprises at least one genetic modification in at least one of the endogenous genes selected from the group consisting of )(ESL RK11.
RPE1, TKL 1, and TALL
1001951 E38, The recombinant yeast cell of Er, wherein the recombinant yeast cell comprises one or more genetic modifications that leads to the overexpression of at least one of the endogenous genes selected from the group consisting of XKS1, RK.I I. RPEI. TKL1, and TALI.
1001961 E39. The recombinant yeast cell of any one of EI-E38, wherein the recombinant yeast cell further comprises a deletion or disruption ()Ione or more aldose reductase genes.
1001971 E40. The recombinant yeast cell of E39, wherein the aldose reductase gene is GRE3 or \TR!.
[001981 E41. The recombinant yeast cell of E40. wherein the recombinant yeast cell comprises a deletion or dismption of GRE3 and Y.PR1.
1001991 E42. The recombinant yeast cell of any one of El-E41. wherein the )east cell further comprises a modification of the endogenous PGM I gene.
1002001 E43. The recombinant yeast cell of E42. wherein the modification of the endogenous PGM1 gene results ia the oycrespression of PGM I.
1002011 E44. The recombinant yeast cell of any one of El-E43. wherein the recombinant yeast cell is capable of growing on sy lose as the sole carbon source.
1002021 E45. A method for producing a fermentation product comprising contacting the recombinant yeast cell of any one of El -E44 with a carbon source, wherein said carbon source comprises xylose and/or sylart.
100.2031 E46. A method for producing a fermentation product comprising contacting the recombinant yeast cell of any one of Et -.E44 with a carbon source, wherein said carbon source comprises ylose.
1002041 E47. The method of E45. wherein the recombinant yeast cell is further grown on a media supplemented with iron.
1002951 E48. The method of E45 or E46, wherein the fermentation product is selected from the group consisting of ethanol, lactic acid, 3-hydroxy-pr-opionic acid, hydrogen. butyric acid, acrylic acid, acetic acid, succinic acid, citric acid. mak acid, fumaric acid, an amino acid, 1.3-propane-did. ethylene, glycerol, acetone. isopropyl alcohol. butanol, a 11-lactaimn, an antibiotic, a eephalosporin, and combinations thereof 1002061 E49. The method of EC/. wherein the fermentation product is ethanol.
[002071 E50. The method of any one of E45-E48, further comprising recovering the .fennentation product.

1002081 E51. A method of producing ethanol comprising contacting a carbon source comprising lose and/or xylan with the recombinant yeast cell of any one of E -E44 in a fermentation medium under conditions wherein ethanol is produced.
1002091 E52. A method of producing ethanol comprising contacting a carbon source comprising xylose with the recombinant yeast cell of an one of El -E44 in a ferinentation medium under conditions Atherein ethanol is produeed.
1002101 E53. The method of E50. wherein the fermentation medium is supplemented with iron.
1002111 E54. The method of E50 or E.51. wherein the carbon source comprises cellulosic or lignocellulosic biomass.
1002121 E.55. The method of E52. wherein the cellulosic or lignocellulosic biomass is selected from the group consisting of insoluble cellulose. crystalline cellulose. pretreated hardwood.
paper sludge. pretreated corn stover.
pretreated sugar cane bagasse. pretreated corn cobs. preheated switchgrass.
pretreated municipal solid waste.
pretreated thsiillcrs dried grains. pretreated wheat straw. COM fiber. agave.
trees. COM stover. wheat straw. sugar cane bagasse. sx% itchgrass. and combinations thereof.
1002131 E56. The method of ED. wherein the biomass is corn stover.
1002141 E37. The method of claim an one of E50-E54. further comprising recovering the ethanol.
1002151 E58. The recombinant yeast cell of am one of E1-E44 for use in a fermentation which convert a carbon source into a fermentation product. wherein said carbon source comprises xylosc and/or xylan.
1002161 E59. The recombinant yeast cell of E15, wherein the recombinant yeast cell comprises heterologons expression of one or mom poly nucleotides encoding XKS1. RIX] I. FtPEl. TKL1.
and/or TALI
1002171 Those skilled in the art will recognize. or be able to ascertain using no more than routine experimentation, main equivalents to the specific embodiments of the invention described herein. Such equivalents arc intended to be encompassed by the following claims.

Claims (10)

. J = =

We Claim:

1. A recombinant yeast cell comprising (a) at least one or more mutations in an endogenous gene encoding a protein associated with iron metabolism; and (b) at least one heterologous gene encoding a polypeptide having xylose isomerase activity;
whereby the at least one or more mutations in an endogenous gene encoding a protein associated with iron metabolism results in an increased ability to utilize xylose by the recombinant yeast cell as compared to a wild-type yeast cell corresponding to the recombinant yeast cell lacking the at least one or more mutations in an endogenous gene encoding a protein associated with iron metabolism, and wherein the endogenous gene encoding a protein associated with iron metabolism is YFH1.

2. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell comprises one or more mutations in the endogenous YFH1 gene that results in a polypeptide comprising a T163P substitution, wherein the position of the substitution is relative to the amino acid positions of SEQ ID NO:31.

3. The recombinant yeast cell of claim 1 or claim 2, wherein the recombinant yeast cell further comprises deletion or disruption of an endogenous gene selected from the group consisting of YAPS and CCC1.

4. The recombinant yeast cell of any one of claims 1 to 3, wherein the heterologous gene (b) encodes a polypeptide having at least 80%, 85%, 90%, 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 35, 37, 39, and 41, the polypeptide having the xylose isomerase activity.

5. The recombinant yeast cell of any one of claims 1 to 4, wherein the recombinant yeast cell comprises one or more genetic modifications that lead to overexpression of at least one endogenous gene selected from the group consisting of XKS1, RKI1, RPE1, TKL1, and TALI..

6. The recombinant yeast cell of any one of claims 1 to 5, wherein the recombinant yeast cell further comprises a deletion or disruption of one or more aldose reductase genes.

7. The recombinant yeast cell of claim 6, wherein the aldose reductase gene is GRE3 or YPR1.

8. The recombinant yeast cell of any one of claims 1 to 7, wherein the yeast cell further comprises a modification of the endogenous PGM I gene, wherein the modification of the endogenous PGM1 gene results in the overexpression of PGM1.

9. A method for producing a fermentation product comprising contacting the recombinant yeast cell of any one of claims 1 to 8 with a carbon source, wherein said carbon source comprises xylose and/or xylan.

10. The recombinant yeast cell of any one of claims 1 to 3, wherein the recombinant yeast cell further comprises heterologous expression of one or more polynucleotides encoding XKS1, RPE1, TKL1, and TALI .